B    3    bS3    7fl7 


I9I<^ 


pis.'' 


#tat?  of  5Rl|05f  UBimh  mh  l^ranihmtt  JlantattottB 


CONTRIBUTIONS 


Bacteriology  of  the  Oyster 


THE  RESULTS  OF  EXPERIMENTS  AND  OBSER- 
VATIONS MADE  WHILE  CONDUCTING 
AN    INVESTIGATION    DIRECTED 
AND    AUTHORIZED    BY  THE 
COMMISSIONERS  OF  SHELL 
FISHERIES     OF    THE 
STATE    OF     RHODE 
ISLAND. 


LESTER  A.   ROUND,  PH.  D. 


PROVIDENCE  : 

E.   L.   FREEMAN   CO.,    STATE   PRINTERS, 
1914 


^tat?  of  EI1O60  Sfilattb  nt\h  '^vomhnui^  JpiautattottH 


CONTRIBUTIONS 


Bacteriology  of  the  Oyster 


THE  RESULTS  OF  EXPERIMENTS  AND  OBSER- 
VATIONS MADE  WHILE  CONDUCTING 
AN    INVESTIGATION     DIRECTED 
AND    AUTHORIZED    BY   THE 
COMMISSIONERS  OF  SHELL 
FISHERIES     OF    THE 
STATE    OF     RHODE 
ISLAND. 


LESTER  A.   ROUND,   PH.  D. 


PROVIDENCE : 

FREEMAN    CO.,    STATE    PRINTERS, 

1914 


PUBLIC 
HEALTH 
LIBRARY 


Kh 


iikBltf"^' 


TABLE  OF  CONTENTS. 


1.  Preface 1 

2.  Bacteriology  of  the  Branchial  and  Cloacal  Chambers  of  the  Oyster 4 

3.  Review  of  Methods  of  Shellfish  Examination 11 

4.  Bacteriology  of  the  Shell  Liquor  and  "Washings"  from  the  Body  of 

the  Oyster 22 

o.     Comparison  of  the  Bacterial  Contents  of  the  Shell  Liquor  and  Stomach 

Contents  of  the  Oyster 46 

6.  Length  of  Tmie  Necessary  for  Bacteria  to  Pass  through  the  Intestinal 

Tract  of  the  Oyster 49 

7.  Bacterial  Content  of  Oysters  During  Storage 51 

8      Cleansing  of  Polluted  Oysters 63 

9.     Ex]3eriments  on  the  Hibernation  of  the  Oyster 78 

10.     Changes  Suggested  in  "Standard  Methods  of  Shellfish  Examination"  .  .  115 

BIOLOGY 

LIBRARY 

G 


PREFACE. 

In  March,  1910,  the  Commissioners  of  Shell  Fisheries  authorized 
an  examination  of  the  sanitary  condition  of  the  waters  of  Narragansett 
Bay  and  its  tributaries,  relative  to  the  growing  of  oysters.  They 
placed  Prof.  F.  P.  Gorham,  head  of  the  Department  of  Bacterio- 
logy of  Brown  University,  in  charge  of  this  work,  with  the  writer  as 
an  assistant. 

On  beginning  this  work  it  was  found  that  there  were  many  prol^lems 
that  would  require  more  study  than  could  be  given  while  performing 
the  routine  bacteriological  examinations  of  water,  mud,  shellfish,  etc. 
To  a  great  extent  this  work  was  new  and  the  method  of  procedure  had 
to  be  worked  out  as  the  investigation  progressed.  While  there  had 
been  much  work  performed  upon  shellfish  examinations,  both  abroad 
and  in  this  country,  there  were  still  many  problems  which  had  not 
been  solved  and  it  was  deemed  advisable  by  the  Commission  that 
some  of  these  problems  which  were  of  great  importance  to  the  shellfish 
industry  should  be  given  special  attention. 

The  writer  was  early  assigned  to  conduct  a  series  of  experiments  and 
investigations  along  the  lines  that  had  been  found  would  apparently 
prove  of  the  greatest  advantage  to  the  oyster  industry.  The  results 
of  some  of  these  investigations  is  published  in  this  booklet. 

The  writer  wishes  to  take  this  opportunity  to  express  his  sincerest 
thanks  to  Prof.  F.  P.  Gorham,  head  of  the  Bepartment  of  Bacterio- 
logy of  Brown  University,  whose  valuable  advice  and  criticisms  have 
been  exceedingly  helpful  and  under  whose  direction  the  work  herein 
reported  has  been  done;  to  Drs.  A.  D.  Mead,  H.  E.  Walter  and  P.  H. 
Mitchell,  who  have  made  valuable  suggestions  and  criticisms  on  differ- 
ent points  in  the  work;  to  the  members  of  the  Narragansett  Bay 
Oyster  Company,  the  American  Oyster  Company,  the  Wickford 
Oyster  Company  and  the  Beacon  Oyster  Company,  ri,n<>l;tq  Captain 
William  B.  Welden,  all  of  whom  have  rendered  valuable 'aid  ili  carrjnng  ' 
out  many  of  the  experiments;  also  to  Mr.  W.  B.  Mason  cf  TlieMec-^. 
chants'  Cold  Storage  and  Warehouse  Company  who'ha.^ 'giVen'fl-ee'' 
use  of  the  company's  cold  storage  rooms  for  the  experiments  on 
hibernation. 

L.  A.  R. 

Brown  University, 
May  1,  1914. 


911A98 


THE     BACTERIOLOGY     OF    THE     CLOACAL     AND     GILLS 
CHAMBERS  OF  THE  OYSTER. 


In  describing  the  anatomy  of  the  gills  of  the  oyster,  Kellogg  in  his 
book  on  "Shellfish  Industries"  makes  the  following  statement: 
"Behind  the  body  the  four  gills  unite  so  as  to  separate  a  space  above 
the  cloacal  chamber,  from  the  large  mantle  chamber  below."  From 
this  statement  it  has  been  assumed  at  times  that  the  two  chambers 
were  entirely  distinct  and  so  constructed  that  bacteria  could  not  pass 
from  one  chamber  to  the  other,  and  that  for  this  reason  the  bacterial 
content  of  the  two  chambers  would  differ.  Anyone  familar  with  the 
anatomy  of  the  oyster  knows  that  every  day  several  gallons  of  water 
are  filtered  through  the  gills  into  the  cloacal  chamber.  While  it  is 
probable  that  most  of  the  protozoa  and  algae  are  caught  in  the  mucus 
which  the  gills  secrete,  it  is  also  probable  that  a  great  many  of  the 
l^acteria  escape,  being  entrapped  by  the  mucus  and  pass  on  into  the 
cloacal  chamber.  But  even  though  the  gill-filter  were  proven  to  be 
bacteria-proof  no  one  has  demonstrated  that  bacteria  cannot  pass  along 
the  space  between  the  mantle  and  the  shell,  or  around  the  edge  of  the 
shell,  between  the  flaps  of  the  mantle  and  so  pass  from  one  chamber 
to  the  other.  While  it  seems  very  probable  from  the  structure  of  the 
oyster  that  bacteria  can  pass  fron  one  chamber  to  the  other  without 
difficulty,  properly  conducted  experiments  are  necessary  to  prove  it. 
In  order  thus  to  prove  that  bacteria  can  and  do  pass,  from  one 
chamber  to.  ^the* other,  the  following  experiments  were  tried. 

Sepibs'J.   ;   c'.^  '  '; 

Exp.  1.  A  well  shaped  mature  oyster  about  four  inches  long  and 
two  broad  was  selected.  Care  was  taken  to  obtain  an  oyster  with  a 
flat  right  valve.  The  oyster  was  placed  in  a  frame  with  the  left 
valve  down  so  that  the  right  valve  was  level  and  was  then  clamped 
firmly  to  the  table.  A  hole  was  bored  through  the  right  valve  into 
the  gill  chamber  quite  close  to  the  edge  and  another  into  the  cloacal 


q   U 


•'fffff^ft*'^ 


BACTERIOLOGY    OF   THE    OYSTER.  6 

chamber,  at  the  anal  orifice.  This  latter  opening  was  ^  to  %  of  an 
inch  above  the  lower  end  of  the  partition  which  separated  the  two 
chambers.  A  loopful  of  B.  prodigiosus  was  then  placed  in  the  gill 
chamber,  and  loopfuls  were  removed  from  the  cloacal  chamber  and 
plated  at  intervals  of  ten  minutes  for  one  hour,  and  then  at  the  end  of 
two  hours  and  three  hours.  B.  prodigiosus  is  a  non-motile  organism 
and  was  chosen  because  of  its  ease  of  identification  and  because  in  all 
our  work  extending  over  four  years  we  have  never  isolated  it  from 
oysters. 

No  red  colonies  were  found  in  the  two  control  samples  from  the 
two  chambers,  but  every  plate  made  from  the  cloacal  chamber  after 
the  introduction  of  the  B.  prodigiosus  into  the  gill  chamber  showed 
colonies  of  B.  prodigiosus. 

Exp.  2.  The  above  experiment  was  repeated  with  another 
oyster  and  B.  prodigiosus  was  again  found  in  the  cloacal  chamber  ten 
minutes  after  its  introduction  into  the  gill  chamber. 

Series  II. 

Exp.  1.  In  another  set  of  experiments  four  oysters  were  used. 
In  these  oysters  five  holes  were  bored  as  indicated  in  the  plate 
shown  on  opposite  page.  Three  of  these  holes  opened  into  the  gill 
chamber  (1,  4,  5).  Another  hole  (2)  was  made  into  the  cloacal 
chamber  near  the  anal  orifice,  and  the  last  hole  (3)  opened  on  the  edge 
of  the  mantle  about  an  inch  above  the  anal  orifice. 

All  four  of  these  oysters  were  inoculated  in  hole  No.  5  with  a  loopful 
of  B.  prodigiosus.  Loopfuls  from  the  other  holes  were  inoculated 
upon  agar  slants  at  two  minute  intervals,  for  ten  minutes  and  then 
every  five  minutes,  for  twenty  minutes,  making  a  total  of  30  minutes 
in  each  case.     The  result  is  seen  in  the  following  talile : 


BACTERIOLOGY    OF    THE    OYSTER. 


Table  No.  1. 


Showing   the  time   at  which   B.  prodigiosus  teas  isolated  from  the 
different  holes  after  inoculation  of  the  gill  chamber  at  hole  No.  5. 


Oyster  No. 


Hole  No. 
Control . 

2  min . . 

4    '••    . . 


4 
0 

+ 
+ 
+ 

+    + 


1  2 

0  0 

0  + 

0  + 

+  + 

+  + 


3 
0 
0 

+      + 

+      + 
+      + 


+  1  + 

+  1  + 


4    1 

o!  0 

0^   0 

+ 
+ 
+ 

+ 
+ 

+ 

+ 


-f-  =  presence  of  B.  prodigiosus.     0  ■=  absence  of  B.  prodigiosus. 


From  this  table  it  is  seen  that  in  oysters  Nos.  1  and  2  B.  prodigiosus 
was  isolated  from  all  the  holes  at  the  end  of  four  minutes;  in  oyster 
No.  3  at  the  end  of  six  minutes,  and  in  oyster  No.  4  not  until  the  end 
of  fifteen  minutes.  Oyster  No.  4  was  a  long  narrow  oyster  and  hole 
No.  3  in  this  case  wfis  necessarily  moved  further  into  the  middle  of 
the  oysters,  so  that  the  opening  came  nearly  over  the  stomach,  and  so 
did  not  reach  the  cloacal  chamber.  In  the  other  cases,  hole  No  3  was 
made  nearer  the  edge  of  the  oyster  and  close  to  the  free  edge  of  the 
mantle,  so  that  there  was  much  greater  chance  of  bacteria  reaching 
the  hole  from  the  liquor  between  the  free  edges  of  the  mantle,  for 
the  edges  of  the  mantle  are  everywhere  free  except,  at  the  "head"  end, 
where  the  the  edges  fuse  and  form  a  hood,  which  is  attached  to  the 
body  by  a  flap  of  tissue.  Between  the  free  edges  of  the  mantle  and 
between  the  hood  and  the  body  there  is  a  space  which  extends  around 
the  whole  oyster  and  forms  a  kind  of  moat  or  trench  filled  with  the 
liquor  in  which  bacteria  can  and  do  move  by  the  currents  set  up  by  the 
ciliary  mfechanism  of  the  oyster,  which  will  be  described  a  little  later. 
It  will  also  be  noticed  that  in  every  oyster  of  this  series  B.  prodigiosus 
was  isolated  from  hole  No.  4  before  they  were  from  hole  No.  1.  although 
the  distance  between  holes  Nos.  4  and  5  were  nearly  twice  as  far  as 


BACTERIOLOGY    OF    THE    OYSTER.  7 

between  Nos.  1  and  5.  It  will  further  be  noted  that  in  oysters  Nos.  1 
and  3  B.  prodigiosus  was  isolated  from  the  cloacal  chamber  (hole  No. 
2)  before  it  was  found  in  hole  No.  1,  although  the  distance  between 
holes  Nos.  5  and  2  was  also  about  twice  as  far  as  between  5  and  1 .  In 
no  case  did  B.  prodigiosus  appear  at  hole  No.  1  before  it  did  at  No.  2. 

Series  III. 

A  third  set  of  experiments  were  now  performed  in  order  to  show 
that  bacteria  can  pass  from  the  cloacal  chamber  to  the  gill  chamber 
and  to  ascertain,  if  possible,  the  avenue  through  which  this  takes 
place.  In  this  experiment  two  oysters  were  used  and  secured  to  the 
bench  in  the  same  manner  as  in  the  previous  experiments.  Three 
holes  were  bored  into  the  branchial  chamber  as  indicated  by  the 
Nos.  1,  5  and  4  in  the  plate.  One  hole  was  bored  into  the 
cloacal  chamber  as  indicated  by  No.  2  in  the  plate.  Control 
inoculations  were  made  as  before  from  these  four  holes.  These 
showed  no  colonies  of  B.  prodigiosus.  A  loopful  of  B.  prodig- 
iosus was  placed  in  hole  No.  2,  and  loopfuls  were  taken  from  holes 
Nos.  1,  5  and  4  at  intervals  of  two  minutes  for  fourteen  minutes. 
The  results  are  shown  in  table  No.  2. 

Table  No.  2. 

Showing  the  time  at  which  B.  prodigiosus  was  recovered  from  holes 
Nos.  1,  5  and  4  after  inoculation  of  the  cloacal  chamber  at  hole  No.  2. 


Oyster  No. 

1 

2 

Hole  No 

1       .5       4 
0       0       0 
0       0       0 
0       0      0 
+       00 
+      +       0 

+    +    + 
+    +    + 

+    +    + 

15       4 

Control 

0       0       0 

2  minutes    . 

+       00 

4        "       

+      +      + 

6        " 

+      +      + 

8        " 

+      +      + 

10        " 

+      +      + 

12        " 

+      +      + 

14        "        

+      +      + 

+  ^  presence  of  B.  prodigios 


0  =  absence  of  B.  prodigiosus. 


8  BACTERIOLOGY    OF    THE    OYSTER. 

From  this  table  it  can  be  seen  that  B.  prodigiosus  was  isolated  from 
all  the  holes  in  oyster  No.  1  at  the  end  of  ten  minutes  and  in  oyster 
No.  2  at  the  end  of  four  minutes.  It  is  further  seen  that  in  oyster 
No.  1  B.  prodigiosus  appeared  first  at  hole  No.  1,  two  minutes 
later  at  hole  No.  5,  and  after  another  interval  of  tw:o  minutes 
at  hole  No.  4.  In  oyster  No.  2  the  bacillus  appeared  first  at 
hole  No.  1  and  two  minutes  later  at  holes  Nos.  5  and  4.  In  neither 
case  did  B.  prodigiosus  appear  at  holes  5  or  4  before  it  appeared  at  hole 
No.  1,  nor  in  either  case  at  hole  No.  4  before  hole  No.  5. 

To  understand  the  reason  for  these  results  a  description  of  the 
ciliar}^  mechanism  of  the  oyster  is  necessary. 

When  one  opens  an  oyster  without  mutilating  it,  there  is  found 
between  the  two  flaps  of  the  mantle  four  folds  of  tissue  which  are  the 
gills.  These  folds  appear  solid,  but  are  really  flaps  folded  back  upon 
themselves  and  attached  by  the  edges  to  the  body  so  that  really  each 
gill  is  V  shaped  in  cross  section  and  the  four  gills  form  a  double  W 
(WW).  With  the  unaided  eye  it  can  be  seen  that  there  are  flne  stria- 
tions  running  verticallj^  across  each  gill.  These  are  the  gill  filaments. 
If  we  examine  these  filaments  with  a  microscope  we  will  see  innumera- 
able  hairs  or  cilia  about  1 -500th  of  an  inch  long  or  less,  waving  vigor- 
ously back  and  forth.  If  we  examine  the  cilia  closely  we  find  that 
they  lash  vigorously  in  one  direction,  recover  themselves  slowly  and 
repeat  the  vigorous  stroke.  The  movement  is  quite  comparable 
to  a  man  rowing  a  boat.  He  pulls  vigorously  in  one  direction, 
recovers  himself  and  repeats  the  stroke.  Now  if  we  consider  the  boat 
fastened  so  that  it  could  not  move,  the  oarsman's  efforts  would  send 
the  water  past  the  boat  instead  of  propelling  the  boat  through  the 
water.  Here  w^e  have  the  exact  condition  in  the  oyster.  As  Brooks 
(The  Oj^ster,  1906)  says,  these  little  hairs  "set  up  a  current  in  the 
water.  Each  one  is  so  small  that  its  individual  effect  is  inconceivably 
minute,  but  the  innumerable  multitude  causes  a  vigorous  circulation 
and  each  one  is  set  at  such  a  position  that  it  drives  the  water  before 
it  from  the  gill  chamber  into  one  of  the  water  pores  and  so  into  one 
of  the  water  tubes  inside  the  gill.  As  these  are  filled  they  overflow 
into  the  cloacal  chamber  and  fill  that."  This  set  of  cilia  are  located 
on  the  edges  of  the  filaments  and  force  the  water  through  the  gills 
from  the  branchial  into  the  cloacal  chamber.  There  is  another  set 
of  cilia  which  wave  in  the  opposite  direction  and  by  means  of  the 
mucus  which  is  secreted  by  the  mucus  cells,  they  collect  and  entangle 
the  micro-organisms  and  carry  them  over  to  the  free  edge  of  the  gill 


BACTERIOLOGY    OF   THE    OYSTER.  y 

where  a  third  set  of  cilia  located  on  the  very  edge  of  the  gill  conveys 
the  entangled  organisms  on  the  mouth.  The  arrangement  of  these 
last  two  sets  of  cilia  can  be  seen  in  the  plate.  In  this  diagram 
the  bent  arrows  show  the  course  of  the  water  through  the  gills  into 
the  cloacal  chamber.  The  straight  arrows  indicate  the  course  of  the 
mucus  and  the  entangled  micro-organisms  to  the  mouth. 

When  the  valves  of  the  oyster  are  open  the  current  induced  by  the 
cilia  is  carried  out  of  the  oyster  between  the  points  "A"  and  "B." 
When  the  valves  of  the  oyster  are  closed,  however,  the  cilia  keep 
waving  as  vigorously  as  before,  because  the  oyster  has  no  control  over 
their  movement,  but  in  this  case  the  current  cannot  pass  out  between 
the  valves  and  we  have  what  might  be  called  a  closed  circulation. 
Instead  of  going  out  between  the  points  ''A"  and  "B,"  as  is  the  case 
when  the  valves  are  open,  the  current  must  neccessarily  return  to  the 
gill  chamber  around  point  "A,"  for  a  study  of  the  currents  induced  by 
the  cilia  and  taking  the  direction  indicated  by  the  arrows  shows  that  no 
other  course  is  possible.  All  the  cilia  of  the  cloacal  chamber  direct 
their  motion  towards  point  "A"  and  "B."  All  the  currents  in  the 
branchial  chamber  are  either  through  the  gills  into  the  cloacal  chamber 
or  along  the  edge  of  the  gills  to  the  mouth.  As  water  is  driven  through 
the  gills  to  the  cloacal  chamber  water  from  the  cloacal  chamber  must 
necessarily  take  its  place.  As  point ''  A  "  is  the  point  of  least  resistance 
the  water  necessarily  passes  from  the  cloacal  chamber  to  the  gill 
chamber  around  that  point  and  further  not  only  is  there  nothing  to 
obstruct  this  current,  but  the  current  induced  by  the  cilia  on  the  edge 
of  the  gills  is  such  that  it  would  draw  the  water  from  the  cloacal 
chamber  into  the  gill  chamber  around  this  point.  Hence  we  see  that 
in  the  oyster  we  have  a  complete  cycle  of  currents  induced  by  ciliary 
motion.  The  result  is  that  all  the  water  in  the  oyster  is  filtered 
through  the  gills  many  times  in  an  hour  and  the  process  is  repeated 
every  few  minutes. 

It  happens  that  when  bacteria  enter  the  gill  or  branchial  chamber, 
two  courses  are  open.  They  may  follow  the  currents  through  the 
gills  into  the  cloacal  chamber  or  they  may  become  entangled  in  the 
mucus  of  the  gills  and  be  conveyed  along  the  edge  of  the  gills  to  the 
mouth.  The  chances  of  a  bacterium  going  in  either  of  these  courses 
are  about  equal  and  if  many  bacteria  are  present  some  may  go  by  one 
course  and  some  by  the  other. 

!  A  study  of  table  No.  1  will  show  that  the  B.  prodigiosus  followed 
both  of  these  courses,  some  were  entangled  in  the  mucus  and  were 

2 


10  BACTERIOLOGY  OF   THE  OYSTER. 

carried  to  the  mouth  (hole  No.  4)  while  others  escaped  the  mucus 
and  passed  through  the  gills  into  the  cloacal  chamber  (hole  No.  2). 
A  further  study  of  table  No.  1  will  show  that  the  bacteria  passed  with 
the  currents  for  this  particular  bacterium  was  non-motile  and  so 
could  not  have  reached  the  different  points  by  its  own  activity. 
Moreover,  the  interval  of  time  which  separated  the  inoculation  of  the 
branchial  chamber  and  the  subsequent  recovery  of  the  bacterium 
from  the  different  holes  in  series  II  was  only  4  minutes  in  all,  except 
two  cases  when  it  was  six  and  fifteen  minutes.  The  distance  between 
holes  5  and  4,  5  and  2,  and  5  and  3,  in  all  cases  was  at  least  an  inch, 
in  most  cases,  more.  In  series  III  the  bacterium  was  recovered  from 
all  the  holes  in  four  minutes  in  one  case  and  ten  minutes  in  the  other . 
The  rate  of  travel  of  bacteria  varies  with  the  species,  temperature, 
etc.,  but  it  is  inconceivable  that  a  bacterium  of  the  speediest  variety 
could  move  a  distance  of  over  an  inch  in  four  minutes  by  its  own  activ- 
ity. In  the  case  in  hand,  i.  e.  a  non-motile  bacterium,  it  is  out  of 
the  question. 

It  is  also  seen  in  table  No.  1  that  in  two  cases  B.  prodigiosus  was 
isolated  from  hole  No.  2  before  it  was  recovered  from  hole  No.  1.  In 
the  two  other  cases  they  were  recovered  at  the  same  time.  While  this 
is  not  conclusive  it  leads  the  writer  to  beiieve  that  the  bacteria 
isolated  at  hole  No.  1  had  previously  passed  through  the  gills  and  the 
cloacal  chamber  and  back  into  the  branchial  chamber  by  the  return 
current.  The  results  of  the  experiments  in  series  III  lend  support  to 
this  view.  The  bacteria  did  not  go  directly  from  hole  5  to  hole  1 
because  the  currents  along  the  edge  of  the  gills  is  too  strong  to  allow 
a  bacterium  to  pass  in  that  direction.  An  examination  of  this  current 
under  the  microscope  will  convince  anyone  that  a  bacterium  could  not 
travel  in  that  direction. 

A  study  of  table  No.  2,  which  shows  the  appearance  of  B.  prodigiosus 
in  the  gill  chamber  after  the  inoculation  of  the  cloacal  chamber, 
shows  that  the  organisms  appeared  at  hole  No.  1  and  later  at  hole  Nos. 
5  and  4.  This  is  the  order  of  time  in  which  a  current  from  the  cloacal 
chamber  and  taking  the  direction  of  the  arrows  of  the  edges  of  the 
gills  would  appear  at  holes  Nos.  1,  5  and  4,  in  the  branchial  chamber. 

From  the  foregoing  facts  it  is  plain  that  the  gills  are  not  bacteria 
proof;  that  bacteria  can  and  do  pass  from  the  gill  chamber  to  the 
cloacal  chamber  through  the  gills  and  moreover,  that  bacteria  may 
pass  from  the  cloacal  chamber  to  the  gill  chamber  without  passing 
through  the  gills.     It  is  seen  that  we  have  a  complete  circle  of  currents 


BACTERIOLOGY  OF  THE  OYSTER.  11 

within  the  closed  sheh  of  the  oyster  which,  under  the  conditions  of  the 
experiments,  makes  a  complete  circuit  several  times  in  an  hour, 
and  thus  ensures  a  thorough  mixing  of  the  water  and  the  bacterial 
content  of  the  two  chambers.  In  the  conditions  of  the  experiments 
the  complete  circuit  was  made  in  at  least  six  minutes  and  in  three 
cases  in  so  short  a  period  as  four  minutes.  It  naturally  follows  that 
any  difference  of  bacterial  count  between  the  two  chambers  is  not  to  be 
expected  and  such  differences  as  are  observed  are  within  the  limits  of 
experimental  error. 

METHODS  OF  SHELLFISH  EXAMINATION. 

As  soon  as  sufficient  epidemiological  evidence  had  been  accumulated 
to  show  conclusively  that  oysters  are  under  certain  circumstances- 
contributing  factors  in  the  spread  of  typhoid,  Asiatic  cholera  and  other 
gastro-enteric  disturbances,  it  was  but  natural  that  bacteriologists 
should  look  for  the  specific  cause  of  these  diseases  in  the  oysters 
themselves.  If  the  typhoid  bacillus  and  the  spirillum  of  Asiatic 
cholera  could  be  found  in  oysters,  that  would  be  evidence  which  no 
one  could  dispute.  Although  diligent  search  has  been  made  for  the 
typhoid  bacillus  in  oysters  on  numerous  occasions  since  1893,  it  is 
interesting  to  note  that  there  are  on  record  four  instances  only  in 
which  B.  typhosus  has  been  reported  to  have  been  isolated  from 
oysters.  The  first  instance  was  reported  by  Klein. '^  Regarding  this 
finding  Klein  says : 

"In  view  of  the  importance  likely  to  be  attached  to  the  finding 
of  this  bacillus  in  such  numbers  in  one  of  these  East  Coast  oysters, 
particular  care  has  been  exercised  in  subjecting  it  to  every  possible 
test  .  .  .  As  a  result,  in  all  and  every  one  of  its  characters  it 
coincides  with  the  typhoid  bacillus  obtained  from  the  spleen  of  a 
typical  case  of  tyi^hoid  fever,  and  for  this  reason  I  am  prepared  to 
affirm  that  this  bacillus  obtained  from  the  "Deep  Sea"  oyster  is  the 
typhoid  bacillus."  Besides  the  cultural  tests  used,  the  Bordet- 
Durham  reaction  (macroscopic  agglutination  with  immune  serum 
1:100)  and  Pfeiffer's  phenomenon  were  also  used  and  both  proved 
positive  while  the  controls  in  both  instances  were  negative.  In 
this  instance  the  evidence  seems  quite  sufficient  to  support  Klein's 
assertion. 

^Report  of  the  Medical  Officer  to  the  Local  Government  Board,  1894-5.     Supplement,  Appendix 
No.  2,  p.  115. 


12  BACTERIOLOGY  OF  THE  OYSTER. 

The  second  instance  is  cited  by  Fuller:^  In  1902  at  a  meet- 
ing of  physicians  at  Pera,  Turkey,  it  was  reported  that  a  large 
percentage  of  the  typhoid  cases  occurring  in  Constantinople  could  be 
traced  to  the  consumption  of  polluted  oysters  and  an  examination 
of  the  oysters  in  a  few  instances  showed  the  presence  of  B.  typhosus. 
The  writer  has  not  been  able  to  obtain  the  reference  to  this  paper  and 
the  characteristics  of  the  species  have  not  been  studied.  As  a  result 
no  definite  comment  can  be  made  upon  the  findings  in  this  instance. 

The  third  instance  is  reported  by  Johnstone.^  There  is  not  so 
good  evidence  to  support  the  identity  of  this  bacillus  as  in  the  case 
reported  by  Klein.  Johnstone's  bacillus  "formed  acid  and  gas  in 
bile  salt  glucose  broth"  and  a  "a  slight  discoloration  in  lactose 
litmus  broth"  and  "agglutinated — in  a  dilution  of  one  to  thirty — 
in  a  serum  which  gave  a  positive  reaction  with  a  known  strain  of 
bacillus  typhosus."  All  authorities  are  agreed  that  the  typhoid 
bacillus  produces  no  gas  in  any  sugar  medium.  In  regard  to  the 
agglutination  in  a  dilution  of  one  to  thirty,  the  writer  is  inclined  to 
question  the  specificity  of  so  low  a  dilution.  The  report  referred  to 
above  does  not  say  what  the  titre  of  the  serum  was  with  any  known 
strain  of  tji^hoid,  nor  whether  one  to  thirty  was  the  highest  dilution 
that  would  give  a  positive  reaction,  though  we  are  led  to  suspect  that 
this  was  the  case.  A  dilution  of  one  to  thirty  cannot  be  relied 
upon  explicitly,  for  other  organisms  closely  related  to  typhoid  as 
some  strains  of  B.  coli  will  agglutinate  in  a  dilution  of  one  to  thirty 
and  in  the  case  of  a  strong  serum  in  one  to  one  hundred.^ 

In  1908  Stiles*  isolated  four  organisms  from  oysters  obtained  from 
Jamaica  Bay,  Long  Island  which  "resembled  B.  typhosus  biologi- 
cally, but  did  not  agglutinate  typhoid  immune  serum."  In  1911, 
while  investigating  an  epidemic  of  typhoid  following  a  banquet  given 
October  5,  1911,  at  the  Music  Hall,  Goshen,  N.  Y.,  Stiles  again 
examined  oysters  from  Jamaica  Bay,  where  the  oysters  were  obtained 
for  the  banquet  and  in  this  instance  he  was  able  to  isolate  two 
strains  of  B.  typhosus  from  oysters  "which  had  been  allowed  to 
'drink'  under  an  oyster  house  at  Inwood,  Long  Island."     Besides 

^The  Distribution  of  Sewage  in  the  waters  of  Narragansett  Bay,  with  Especial  Reference  to  the 
Contamination  of  the  Oyster  Beds,  App.  to  Rep.  of  Commissioner  of  Fisheries  for  year  ending 
June  30,  1901. 

^Routine  methods  of  Shellfish  Examination  with  Reference  to  Sewage  Pollution,  Journal  of 
Hygiene,  IX.  1909,  433. 

^Hiss  &  Zinsser;  Text  Book  of  Bacteriology,  1912,  p.  42. 

^Bureau  of  Chemistry,  Bulletin  No.  136. 


BACTERIOLOGY  OF  THE  OYSTER.  13 

showing  all  the  cultural  characteristics  of  the  typhoid  bacillus,  it 
also  agglutinated  in  five  minutes  in  a  1:1000  dilution  of  typhoid 
immune  serum.  This  organism  was  isolated  from  the  oysters  seven 
days  after  they  were  taken  from  the  water.  Later  oysters  from  the 
same  lot  were  examined  after  they  had  been  out  of  the  water  twenty- 
one  days  and  kept  at  39°  F.  An  organism  was  isolated  which 
resembled  typhoid  in  all  its  cultural  characteristics  and  agglutinated 
macroscopically  in  a  chlution  of  1 :1000.  This  test  was  confirmed  by 
hanging  drop  preparations  in  dilutions  of  1 :  200. 

There  can  be  no  possible  doubt  that  the  organisms  isolated  by  Stiles 
are  true  typhoid  bacilli,  while  little  can  be  desired  to  confirm  the 
identity  of  the  organism  isolated  by  Klein. 

An  interesting  feature  of  the  work  of  Stiles  is  that  he  demon- 
strated the  typhoid  bacillus  in  oysters  which  had  been  infected 
under  natural  condition  and  which  had  been  kept  out  of  water 
for  three  weeks.  Klein, ^  Foote^  Herdman  and  Boyce,^  and 
others  have  reported  instances  in  which  typhoid  bacilli  have  been 
isolated  after  varying  lengths  of  time  up  to  18  days  after  infection 
from  oysters  artificially  infected  with  large  numbers  of  typhoid  bacilli 
in  pure  cultures  or  from  typhoid  stools  and  kept  in  sea  water  in  the 
laboratory.  So  far  as  the  writer  is  aware  Stiles  is  the  first  one  to  show 
that  oysters  infected  under  normal  circumstances  with  sewage  con- 
taining typhoid  bacilli  and  kept  under  favorable  conditions  can  still 
harbor  B.  typhosus  after  21  days.  The  condition  here  are  somewhat 
different  from  laboratory  experiments  in  that  in  sewage  along  with  the 
typhoid  bacilli  are  other  bacteria  whose  influence  is  exceedingly 
hostile  to  the  growth  of   B.  typhosus.* 

It  is  interesting  to  see  that  this  organism  has  been  isolated  so  few 
times,  in  spite  of  the  abundant  epidemological  evidence  in  so  many 
instances  which  points  conclusively  to  the  infection  of  oysters  and 
other  shellfish  with  typhoid  bacilli.  The  reason  for  this,  however, 
is  quite  readily  understandable  when  we  consider  the  number  of 
typhoid  bacilli  which  could  be  found  in  the  sewage  of  any  town  or 
city  in  comparison  with  the  number  of  other  organisms  found  in 
that  same  sewage.     It  would  be  a  case  of  searching  for  the  proverbial 

^Loc.  cit. 

^A  Bacteriological  study  of  Oysters,  with  Special  Reference  to  them  as  a  source  of  Typhoid 
Infection,"  18th  Ann.  Report  Com.  State  Board  of  Health. 

'Oysters  and  Disease,  Thompson  Yates  Laboratory  Report,  1-2. 
^Jordan,  Russell  &  Zeit,  Journal  of  Infectious  Diseases  1,  1904,  641. 


14  BACTERIOLOGY  OF  THE  OYSTER. 

needle  in  the  hay  stack.  Moreover  the  incubation  period  of  typhoid 
varies  from  two  to  three  weeks  and  this  would  make  the  period  of 
infection  two  or  three  weeks  before  suspicion  would  be  thrown  on  the 
oysters.  That  is,  two  or  three  weeks  would  elapse  after  infection, 
before  we  began  to  look  for  the  organism.  During  this  space  of  time 
the  other  oysters  of  the  same  laying  would,  in  all  probability,  have 
time  to  rid  themselves  of  the  organisms,  provided  they  too  were 
infected.  In  the  case  of  an  epidemic  of  typhoid  due  to  eating  raw 
oysters,  the  time  to  examine  the  oysters  for  typhoid  bacilli  would  be 
at  the  moment  they  were  eaten.  We  may  be  quite  sure  from  the 
history  of  the  cases  that  the  oysters  which  were  consumed  did 
contain  B.  typhosus,  but  we  have  no  assurance  that  all  the  oysters 
of  that  particular  bed  contained  the  organism.  There  is  great 
variation  in  the  number  of  sewage  organisms  contained  in  the  indi- 
vidual oysters  of  the  same  bed.  This  individual  variation  will  be  still 
greater  if  the  bed  is  large  and  the  amount  of  sewage  small,  tho  highly 
infected  with  B.  typhosus  and  other  sewage  organisms.  Sewage 
does  not  ordinarily  contain  typhoid  bacilli  in  constant  numbers  at 
any  time  and  unless  there  is  an  extensive  epidemic,  B.  typhosus  would 
appear  only  intermittently  and  then  in  comparatively  small  numbers. 
In  view  of  these  facts  the  wonder  is,  considering  that  B.  typhosus 
die  off  rapidly,  both  in  sea  water  and  in  oysters,  that  typhoid  bacilli 
have  ever  been  found  at  all. 

The  spread  of  cholera  through  infected  oysters  has  not  attached  so 
much  attention  as  the  transmission  of  typhoid.  The  latter  is  distribu- 
ted much  more  widely  throughout  the  world  and  the  opportunity 
for  such  transmission  is  much  greater.  Occasionally,  however,  there 
has  appeared  references  to  the  spread  of  cholera  through  infected 
oysters.  In  1849  there  was  a  small  epidemic  of  cholera  in  England 
which  was  attributed  to  eating  oysters.  In  1893  Sir  Richard  Thorne 
attributed  a  number  of  scattered  cases  of  cholera  in  England  to  the 
consumption  of  oysters.  Recently  it  has  been  reported  that  a  large 
extent  of  oyster  beds  in  Italy  have  been  destroyed  because  they  were 
thought  to  be  a  menace  to  the  public  health  on  account  of  the  danger 
of  the  cholera  infection. 

In  most,  if  not  all  epidemics  of  typhoid  from  infected  oysters  or 
other  articles  of  food,  there  have  been  a  greater  number  of  cases  of 
gastro    intestinal    disturbances    which    have    not    developed    into 


BACTERIOLOGY  OF  THE  OYSTER.  15 

typhoid.^  We  cannot  tell  the  exact  cause  of  these  intestinal 
upsets.  It  may  be  due  to  bacteria  other  than  typhoid  or  it  may  be 
due  to  chemical  or  ptomaine  poisons  which  appear  in  the  sewage  as 
the  end  products  of  bacterial  metabolism.  Whatever  the  cause  we 
are  led  to  expect  these  disturbances  as  concomitants  of  any  outbreak 
of  typhoid  due  to  an  infected  food. 

Since  one  can  rely  so  little  upon  the  finding  of  the  specific  disease 
organism  in  sewage  and  in  oysters,  it  was  but  natural  that  an  index 
of  greater  reliability  should  be  sought.  Klein^  at  the  begin- 
ning of  his  experimental  work  as  well  as  in  some  previous  investigations 
ascertained  that  B.  coli  and  other  intestinal  bacteria  form  no  part 
of  the  flora  of  oysters  grown  in  non-polluted  water  and  for  this  reason 
used  B.  coli  as  an  index  of  pollution,  Klein's  observations  in  regard 
to  the  bacterial  content  of  oysters  grown  in  water  free  from  sewage 
has  been  confirmed  by  Houston,^  Ferguson,^  Fuller^  and  others.  The 
presence  of  B.  coli  as  an  indication  of  sewage  pollution  has  been 
adopted  by  all  workers  in  this  field  and  is  the  index  used  to-day  to 
determine  bacteriologically  the  presence  of  fecal  matter. 

In  examining  oysters,  however,  we  have  quite  a  different  problem 
from  the  examination  of  water,  for  we  have  not  only  the  juice,  but  the 
body  of  the  oyster,  the  mucus  covering  the  body,  the  alimentary 
canal,  etc.,  to  consider.  It  is  interesting  to  see  how  the  methods  of 
examination  have  changed  as  our  knowledge  of  the  bacteriology^  of 
the  different  parts  of  the  oyster  has  increased. 

Perhaps  the  first  person  to  make  an  extended  study  of  the  bacteri- 
ology of  the  oyster  was  Klein,  who  in  1893,®  made  a  study  of  the 
"Relation  of  Oysters  and  Disease"  for  the  Local  Government  Board. 
Klein  describes  his  method  of  analysis  as  follows: — 

"Each  oyster  was  carefully  washed  and  brushed  in  a  small  quantity 
of  sterile  water,  with  a  view  to  collect  therein  any  microbes  adhering 
to  its  shell.  Next,  the  oyster,  after  a  further  cleansing  under  a  water 
tap  and  drying  with  a  clean  cloth  was  opened  with  a  sterile  knife. 

'■As  an  illustration,  the  reader  is  referred  to  the  following  reports:  H.  T.  Bulstrode,  in  local 
Government  Board,  32d  Annual  Report,  1902-1903,  Suppl.  App.  A.  pp.  129-189;  H.  W.  Conn,  The 
"Oyster  Epidemic"  of  typhoid  fever  at  Wesleyan  University,  Medical  Record,  46,1894,  743-6; 
G.  W.  Stiles,  Sewage  Polluted  Oysters  as  a  Cause  of  Typhoid  and  other  Gastro-intestina!  Disturb- 
ances, Bureau  of  Chemistry,  Bulletin  136, 1912. 

^Loc.  cit. 

^4^h  Rep.  Royal  Sewage  Commission,  1904. 

■•Bull.  Virginia  State  Board  of  Health,  May,  1909. 

^ILoc.  cit. 

^Supplement  to  Report  of  Medical  Officer  to  Local  Government  Board,  Appendix  No.  2,  pp.  109 
and  117. 


16  BACTERIOLOGY  OF  THE  OYSTER. 

and  its  body  mashed  up  with  the  Hquor  contained  in  the  shell  . 
and  about  34  to  3^  c.c.  of  the  liquor  and  the  oyster  tissue  was  removed 
l3y  means  of  a  freshly  made  capillary  pipette  and  introduced  into  a 
phenolated  broth  tube  which  was  incubated  at  37°  C  for  24  hours." 
If  growth  occurred  the  culture  was  plated  and  the  suspicious  colonies 
fished  and  studied  in  pure  culture.  This  method  allowed  no  compari- 
son between  the  bacterial  content  of  the  shell  liquor  and  the  "oyster 
tissue."  Besides  it  did  not  allow  a  determination  of  the  number  of 
colon  bacilli  in  the  whole  oyster  nor  per  unit  volume.  Moreover, 
we  have  no  evidence  that  any  part  of  the  oyster  tissue  except  the 
epithelium  of  the  outside  of  the  body  and  the  lining  of  the  alimentary 
tract  contain  bacteria  and  this  large  amount  (in  comparison  to  the 
amount  of  shell  liquor)  of  finely  divided  tissue — for  it  must  have  been 
finely  divided  to  have  been  taken  up  in  a  capillary  pipette — would 
interfere  greatly,  if  one  tried  to  obtain  an  accurate  count. 

Chantemesse,  in  June,  1896,  reported  to  the  Academic  de  Medicine, 
Paris,  his  observations  on  the  relation  of  oysters  to  disease.  In  the 
article  presented  at  this  meeting  he  does  not  give  the  details  of  his 
technique,  but  says  the  shell  liquor  and  the  bodies  of  the  oysters  were 
submitted  to  a  bacteriological  examination  and  B.  coli  w^ere  found. 

The  next  important  investigation  after  that  of  Klein  is  the  work  of 
Herdmann  and  Boyce.^  A  great  number  of  experiments  were  per- 
formed on  the  chemistry  and  biology  and  also  on  the  bacteriology 
of  the  oyster.  Only  a  small  part  of  their  work  related  to  the  presence 
of  B.  coli  in  normal  oysters.  For  this  work  the  stomach  contents 
were  used.     The  following  is  quoted  from  their  report : — • 

"The  method  of  analysis  consisted  in  first  cauterizing  the  mantle 
over  the  region  of  the  stomach  and  then  inserting  a  fine  sterilized 
glass  pipette,  the  pipette  was  moved  about  and  when  sufficient  of 
the  contents  of  the  stomach  and  the  juice  had  risen  in  the  pipette, 
the  latter  was  removed  and  its  contents  transferred  to  liquified  agar, 
ordinary  gelatine  or  sea-water  gelatine  and  plate  cultivations  made." 

Apparently  no  attempt  was  made  to  determine  the  number  of  colon 
bacilli  either  per  unit  quantity  or  in  the  contents  of  the  stomach  as  a 
whole. 

The  next  important  investigation  we  have  noted  is  the  work  of  Dr. 
Houston.^     Dr.  Houston's  method  of  analysis  is  as  folloAvs: — 

Kl)  Lancashire  Sea  Fisheries  Memoir  No.  1.  (2)  Proceedings  of  Royal  Society,  1899.  (3) 
Thompson  Yates  Lab.  Rpt.  1-2. 

^Fourth  Report  of  Royal  Sewage  Commission.     Vol.  Ill,  1904 


BACTERIOLOGY    OF   THE    OYSTER.  17 

Cleaning  of  Oysters: — 

"The  outside  of  the  oj^ster  shells  was  Avell  scrubbed  with  soap  and 
water,  and  cleansed  as  thoroughly  as  possible  under  running  water; 
the  shells  were  then  well  washed  in  running  main  water,  and  finally 
with  sterile  water. 

Cleansing  of  the  Hands : — 

"The  hands  of  the  experimenter  were  thoroughly  cleansed  with  a 
hard  scrubbing  brush,  soap  and  water,  then  rinsed  first  with  1:1000 
corrosive  sublimate  solution,  and  finally  with  sterile  water. 

Subsequent  Procedure : — 

"The  03'sters  were  laid  out  upon  a  sterile  towel,  the  flat  shell 
uppermost.  They  were  opened  in  this  position  with  a  sterile  knife, 
held  in  the  right  hand,  while  they  were  held  in  this  position  with  a 
corner  of  the  sterile  towel  grasped  in  the  left  hand.  Great  care  was 
taken  to  avoid  any  loss  of  liquor  in  the  shell.  This  liquor  was  poured 
into  a  sterile  100  c.  c.  cylinder,  the  oyster  was  then  partly  cut  with 
sterile  scissors  and  the  liquor  thus  freed  allowed  to  run  into  the 
cylinder.  Ten  oysters  were  thus  treated  in  each  experiment.  The 
volume  of  the  oyster  plus  the  oyster  liquor  was  read  off,  and  usually 
varied  bew^een  80  and  120  c.c.  so  that  the  oysters,  being  of  medium 
size  and  containing  a  medium  amount  of  liquor,  100  c.c.  might  be 
considered  a  fair  average  of  the  total  shell  contents  of  the  ten  oysters. 
Sterile  water  was  then  poured  into  the  cylinder  up  to  the  1,000  c.c. 
mark,  and  the  whole  well  stirred  with  a  sterile  rod. 

"An  Alternative  Quantative  Method  for  the  Bacteriological 
Examination  of  Oysters. 

''An  alternative  method  for  the  bacteriological  examination  of 
oysters  may  be  given  here,  although  the  routine  work,  except  where 
otherwise  stated,  has  been  carried  out  by  the  foregoing  method. 

"The  oysters  are  cleansed  and  opened,  with  the  same  precautions 
already  noted.  Then  the  body  of  the  oyster  is  cut  into  small  pieces 
with  sterile  scissors :  this  process  should  be  carried  out  in  such  a  way 
as  to  insure  the  thorough  mixture  of  the  gastric  juice  of  the  oyster 
and  the  liquor.  The  oyster,  meanwhile,  is  carefully  held  with  the 
concave  shell  do"\vnwards  and  the  flat  shell  bent  back  or  altogether 
removed.     To  examine  the  liquid  contents  of  the  shell  without  this 


18  BACTERIOLOGY   OF   THE    OYSTER. 

preliminary  step  may  partake  of  the  nature  of  the  examination  of 
the  last  sample  of  sea  water  imbibed  by  the  oyster  before  finally 
closin  g  the  shell.  Indeed,  the  experiments  detailed  elsewhere  seem 
to  indicate  that  per  unit  volume  the  gastric  juice  of  the  oyster  may  be 
more  impure  bacteriologically  than  the  oyster  hquor. 

"  For  cultural  purposes  the  following  quantities  were  made  by  proper 
dilutions:— 100  c.c,  10  c.c,  1  c.c.    1-10  c.c,  1-100  c.c,  1-1000  c.c." 

It  appears  that  this  was  the  first  attempt  to  determine  the  number 
of  B.  coil  or  coli-like  organisms  within  the  oyster.  The  supposition 
was  that  the  supernatant  liquid  above  the  oysters  contained  in  an 
even  distribution  all  the  bacteria  that  were  present  in  the  shell  liquor, 
the  juices  of  the  body,  and  on  the  outside  of  the  oyster.  Whether 
this  assumption  is  true  or  not  will  be  discussed  later  when  the  writer 
takes  up  his  own  experiments.  Houston  also  performed  "a  series  of 
experiments  to  ascertain  the  relation  between  the  biological  (bacterio- 
logical L.  A.  R.)  composition  of  (1)  the  shell  liquor  and  surface 
"washings"  of  the  oyster,  and  (2)  the  "washed  bodies  of  the  oysters." 
In  this  series  of  experiments,  four  in  number,  by  rapid  fire  calculation 
and  assumptions,  Houston  arrives  at  some  very  startling  conclusions.^ 
From  these  experiments  he  states  that  volume  for  volume  the  stomach 
of  the  oyster  contains  more  bacteria  than  any  other  part  of  the 
oyster.  The  method  of  conducting  the  experiments  and  the  premises 
assumed  and  conclusions  drawn  will  be  discussed  more  at  length 
when  the  writer  takes  up  similar  experiments  of  his  own. 

Fuller  in  the  article  cited  above  describes  his  method  as  follows: — 
"In  the  examination,  inoculations  were  made  from  the  liquor 
contained  between  the  shells,  from  the  contents  of  the  intestines, 
stomach,  and  rectum,  and  in  some  cases  from  portions  of  the  visceral 
mass.  In  order  to  obtain  samples  of  the  juice  from  an  oyster  under 
aseptic  conditions,  the  speciments  to  be  examined  were  scrubbed 
thoroughly  in  tap  water  with  a  stiff  brush,  washed  off  in  running 
sterile  water,  and  dried  on  a  sterile  towel,  after  which  they  were 
opened  with  a  sterile  knife.  To  obtain  cultures  from  the  stomach, 
the  top  of  the  mantle  covering  the  interior  end  of  the  oyster  was  slit 
open  and  the  large  palps  on  either  dide  of  the  mouth  pushed  aside; 
the  mouth  region  was  sterilized  by  passing  a  hot  scalpel  over  these 
parts  and  a  portion  of  the  stomach  contents  was  drawn  out  by  means 
of  a  fine  pipette  or  platinum  loop  introduced  through  the  mouth 
opening.     Cultures  from  the  intestines  were  made  in  the  following 


BACTERIOLOGY    OF    THE    OYSTER.  19 

manner:  After  opening  the  shell,  the  oyster  was  removed  from  the 
shell  and  dried  between  filter  papers.  A  hot  spatula  was  then  passed 
upon  the  surface  of  the  mollusk  directly  over  that  portion  of  the  in- 
testine which  it  was  desired  to  reach,  and  the  tube  was  then  opened 
with  a  sterile  scalpel.  Through  this  opening  a  portion  of  the  contents 
was  drawn  out  by  means  of  a  pipette  or  platinum  loop.  Portions 
of  the  visceral  mass  were  obtained  by  cutting  out  cubes  of  flesh  from 
that  portion  of  the  body  after  sterilizing  the  surface  with  a  hot 
scalpel." 

McWeeney^  in  his  examination  of  oysters  on  the  Irish  Coast  used 
the  shell  liquor  alone,  if  abundant.  But  in  cases  where  the  amount 
of  shell  liquor  was  small  he  supplemented  the  small  quantity  of 
liquid  "with  a  block  of  tissue  cut  from  the  animal  itself  so  as  to 
include  portion  of  the  alimentary  canal. " 

The  next  worker  to  do  a  great  deal  of  routine  and  experimental 
work  in  the  examination  of  shellfish  was  H.  W.  Clark.  In  a  prelimi- 
nary report  published  in  1902^  Clark  describes  his  method  of  analysis 
as  follows : — 

"To  determine  the  presence  of  B.  coli  in  the  juice  on  the  shell,  the 
clams,  oysters,  etc.,  were  washed  with  sterilie  water,  then  opened, 
and  this  juice  inoculated  into  bouillon." 

"To  determine  whether  the  germ  was  present  in  the  bodies  of  the 
clams,  oysters,  etc.,  they  were  opened  after  washing  with  sterile 
water,  and  the  intestine,  after  maceration  with  sterile  water,  was 
inoculated  into  phenol  dextrose  bouillon. 

In  1905,  Clark^  in  a  report  covering  his  experimental  work  for  the 
previous  five  and  one-half  years  makes  the  following  statement  in 
regard  to  the  "Examination  of  Raw  Oysters:" — "The  shelUiquor 
and  the  crushed  body  of  the  oyster  were  examined  together  by  insert- 
ing the  entire  mass  in  a  fermentation  tube,  and  if  fermentation  was 
obtained,  carrying  out  the  cultural  tests. " 

In  determining  the  presence  of  B.  coli  in  the  body  of  the  oyster  as 
detailed  in  his  first  report  it  appears  that  Clark  disected  out  the  ali- 
mentary tract.  This  is  not  stated  as  part  of  the  procedure,  but  it  is 
implied  from  the  above  quotation.  This  procedure  would  be  rather 
cumbersome  if  one  attempted  to  use  it  on  a  large  scale  in  routine  exam- 

^Report  on  the  Bacte.ioscopic  Examination  of  Samples  taken  from  Shellfish  Layings  on  the  Irish 
Coast,  Local  Government  Board  for  Ireland,  1904 
^Senate  Document  336,  State  of  Mass.,  1902. 
^Report  Mass.  State  Board  of  Heaith,  1905,  427. 


20  BACTERIOLOGY  OF  THE  OYSTER. 

illations.  Moreover,  Clark  apparently  assumes  that  the  bacteria 
isolated  in  this  manner  all  came  from  the  intestinal  tract  and  that  no 
contaminating  organisms  from  the  mucus  on  the  outside  of  the  body 
entered  into  the  bacterial  flora  of  the  macerated  intestine.  The  writer 
in  some  experiments  to  be  given  in  detail  later  has  shown  that  on  the 
average  there  are  more — often  many  times  more — bacteria  in  the 
mucus  on  the  body  of  the  oyster  than  in  the  total  amount  of  shell 
liquor  and  further  that  volume  for  volume  the  contents  of  the  stomach 
do  not  contain  so  many  bacteria  as  the  shell  liquor.  Since  the  stomach 
contains  more  liquid  on  the  whole  than  the  rest  of  the  intestinal  tract, 
it  is  but  natural  that  it  should  contain  more  B.  coli  than  the  remainder 
of  the  intestinal  tract.  This  would  be  all  the  more  evident  when  it  is 
understood  that  B.  coli  do  not  grow  in  oysters,  but  probably  diminish 
as  they  pass  through  the  intestinal  tract. ^ 

In  his  second  article  cited  above,  the  whole  contents  of  the  oyster 
shell,  "the  shell  water  and  the  crushed  body  of  the  oyster  were  ex- 
amined together  by  inserting  the  entire  mass  in  a  fermentation  tube. " 
Obviously  this  would  allow  of  no  comparison  between  the  bacterial 
flora  of  the  shell  liquor  and  the  body  of  the  oyster.  Yet  in  a  following 
paragraph  and  also  in  a  table  he  gives  the  results  of  the  analysis  in 
"Percent,  of  Samples  Giving  Positive  Tests,"  in  "Shell  Water, 
Intestine"  and  "Mash."  Obviously  there  is  some  discrepancy,  for 
if  he  followed  out  the  method  described  it  would  be  impossible  to 
make  such  a  differentiation.  It  is  possible,  however,  that  Clark  was 
using  a  combination  of  the  technique  as  stated  in  the  two  reports. 
The  shell  water  and  the  "intestinal  content"  were  examined  as  stated 
in  his  report  of  1902,  and  his  "mash"  consisted  of  the  shell  liquor 
and  crushed  body,  the  entire  mass  of  which  was  inserted  into  the  fer- 
mentation tube.  It  would  appear,  however,  that  in  order  to  carry 
out  a  combination  of  these  two  pieces  of  technique,  two  oysters  would 
be  necessary,  one  for  the  shell  liquor  and  intestine  and  another  for 
the  "shell  water  and  the  crushed  body."  If  this  were  true  the 
individual  variation  of  course,  would  allow  of  no  definite  comparison 
between  all  the  parts  tested.  It  may  mean  that  the  remains  of  the 
body  tissue  after  dissecting  out  the  intestine  and  the  unused  portion 
of  the  shell  liquor  were  mixed  and  constituted  the  shell  water  and 
crushed  body.  But,  in  whatever  manner  we  try  to  explain  the  matter , 
the  fact  remains  that  the  method  as  described  is  insufficient  to  account 

^Hardman  &  Boyoc,  loc.  cit. 


BACTERIOLOGY  OF  THE  OYSTER.  21 

for  the  results  obtained.  But,  as  the  results  are  expressed  in  the  text 
and  again  in  more  detail  in  a  table,  we  can  feel  quite  certain  that  the 
method  of  analysis  in  the  second  report  is  not  given  in  sufficient  detail 
and  the  results  expressed  in  the  table  are  accurate  so  far  as  his 
methods  would  allow. 

From  the  table  referred  to  above  it  is  seen  that  in  the  examination 
of  one  hundred  and  forty-five  oysters  approximately  fifty  per.  cent  of 
them  gave  positive  tests  for  B.  coli  in  the  shell  liquor,  seventeen 
per  cent  in  the  "mash"  and  between  seven  and  eight  per  cent,  in 
intestine. 

In  three  following  tables  is  given  the  results  of  the  analysis  of  shell 
liquor  and  intestine  of  265  other  oysters,  making  a  total  of  410  oysters 
examined  in  all.  A  comparison  of  the  percentage  of  positive  results 
in  the  shell  liquor  and  intestine  shows  that  B.  coli  were  found  nearly 
four  times — 50  to  14 —  as  often  in  the  shell  liquor  as  in  the  intestine. 
From  these  experiments  it  seems  apparently  beyond  question  that 
the  greatest  number  of  B.  coli  are  in  the  shell  liquor  of  the  oyster 
and  that  the  body  of  the  oyster  should  be  disregarded  in  our  search 
for  the  colon  bacillus. 

Stiles^  describes  his  method  of  analysis  as  follows : 

"The  examination  of  composite  samples  of  five  or  more  oysters  was 
supplemented  by  inoculating  media  with  the  liquor  from  single  oysters 
to  determine  the  presence  of  Bacillus  coli  in  each.  It  was  also  decided 
to  use  only  the  liquor  bathing  the  oysters,  instead  of  both  meat  and 
liquor,  as  the  latter  represents  the  character  of  the  whole  contents 
of  the  shell  sufficiently  well  to  determine  the  presence  of  pollution." 

Gage^  discribes  his  methods  as  follows : — 

"The  upper  shell  being  removed,  a  portion  of  the  liquor  in  the 
lower  shell  is  now  transferred  to  a  fermentation  tube  with  a  sterile 
pipette,  or  a  portion  of  this  shell-water  may  be  carefully  poured 
directly  from  the  shell  into  the  tube.  The  latter  method  is  much 
simpler  than  the  use  of  pipettes,  but  requires  that  the  shell  be  so 
handled  in  the  previous  operation  that  the  lip  over  which  the  liquor 
is  poured  has  not  been  contaminated.  The  body  is  now  washed  with 
sterile  water,  then  while  held  with  the  fingers  of  the  left  hand,  an 
incision  is  made  with  a  sterile  scalpel  and  a  portion  of  the  intestine 

^Shellfish  Contamination  from  Sewage-Polluted  Waters  and  from  other  Sources,  Bureau  of 
Chemistry,  Bulletin  136,  April  11,  1911. 

^Methods  of  Testing  Shellfish  for  Pollution,  Jour,  or  Infectious  Deseases,  1910,  VII,  7S. 


22  BACTERIOLOGY  OF  THE  OYSTER. 

transferred  with  sterile  forceps  to  another  fermentation  tube,  care 
being  taken  not  to  touch  the  parts  where  the  incision  is  made  with 
the  fingers  or  to  contaminate  it  in  any  way.  This  procedure  i& 
repeated  until  10  individuals  have  been  tested  from  each  sample  jar. " 

It  would  appear  that  the  work  of  Clark  has  had  wide  influence  in 
determining  the  method  of  shellfish  analysis  now  in  use  in  this  country. 
So  far  as  the  writer  is  aware  and  so  far  as  the  literature  at  hand  shows, 
the  only  part  of  the  oyster  used  for  bacteriological  analysis  for  some 
years  has  been  the  shell  liquor.  The  "Committee  on  Standard 
Methods  of  Shellfish  examination"  appointed  by  the  American 
Public  Health  Association  has  recommended  the  use  of  the  shell 
liquor  only.  So  far  as  a  perusal  of  the  recent  literature  is  concerned 
no  one  has  questioned  the  advisability  and  propriety  of  using  the  shell 
liquor  alone  for  analytical  purposes  except  Gorham^  upon  results 
obtained  by  the  writer  in  the  laboratory  of  Brown  University. 

It  will  be  noticed  in  all  the  work  cited  in  which  parts  of  the  intestine 
have  been  used  for  analysis,  except  in  the  case  of  Fuller,  no  mention 
has  been  made  of  trying  to  avoid  taking  bacteria  from  the  outside 
of  the  oyster  as  well.  In  the  writer's  opinion  a  great  many  of  the 
bacteria  alleged  to  have  been  found  in  the  intestinal  tract  have  come 
from  the  mucus  on  the  outside  of  the  body.  There  is  no  doubt  that 
the  intestine  of  the  oyster  does  contain  bacteria  of  sewage  origin, 
but  the  mucus  on  the  outside  of  the  bod}^  is  much  more  likely  to 
contain  such  bacteria. 

BACTERIOLOGY  OF  THE  SHELL  LIQUOR  AND  ^^WASHINGS'^ 
FROM  THE  BODY  OF  THE  OYSTER. 

A  matter  of  great  interest  to  the  writer  is  that  in  all  the  work  done 
upon  oysters  experimentally  and  otherwise  no  one  has  mentioned  the 
mucus  of  the  oyster  or  apparently  realized  that  it  plays  any  part  in  the 
bacteriology  of  the  oyster. 

The  matter  of  the  mucus  in  the  oyster  juice  and  on  the  oyster's  body 
appears  so  self-evident  that  it  seems  impossible  that  it  should  have 
been  entirely  neglected.  This  mucus  serves  at  least  two  purposes. 
(1)  It  acts  as  a  protection  to  the  body  of  the  oyster  and  protects  it 
from  the  deleterious  effects  of  sea  water  in  just  the  same  way  as  the 
mucus  of  the  dog  fish  and  other  selachians  protects  their  skin  from 

^Report  of  Commissioners  of  Shell  Fisheries,  State  of  R.  I.,  1914. 


BACTERIOLOGY  OF  THE  OYSTER.  23 

the  action  of  the  sea  water.  (2)  The  other  and  more  important 
function  from  the  bacteriological  point  of  view  is  that  it  serves  as  a 
net  for  the  entrapping  of  the  food  of  the  oyster  which  consists  largely 
of  diatoms  and  algae,  but  is  made  up  of  all  sorts  of  microscopic 
particles,  living  or  dead,  organic  or  inorganic.  As  a  consequence,  the 
bacteria  as  well  as  the  other  microscopic  organisms  get  entangled  in 
this  mucus. 

AVhen  one  opens  an  oj^ster  and  collects  the  juice,  usually  a  great 
many  particles  of  mucus,  some  particles  very  large  comparatively 
speaking,  are  seen  in  the  liquid.  If  one  handles  an  oyster  after 
opening,  it  is  found  covered  with  a  vicid,  slimy  substance  which  does 
not  wash  off  the  hands  easily  If  the  bodies  of  the  opened  oysters 
are  allowed  to  stand  for  sometime  there  rises  to  the  surface  long 
strings  and  flakes  of  this  greenish  yellow  mucus.  In  shucking  houses 
it  is  customary  to  allow  opened  oysters  to  lie  for  some  time  in  large 
vats  filled  with  water  and  with  occasional  stirring  allow  the  mucus 
to  rise  to  the  surface  of  the  water  and  run  over  the  edge,  if  running 
water  is  used,  or  if  not,  it  is  skimmed  off  with  a  perforated  dipper. 
This  mucus  often  collects  in  "ropes"  two,  three,  or  more  inches  long 
and  sometimes  in  large  flakes  the  size  of  a  half  dollar. 

If  one  examines  the  liquor  of  the  oyster  he  has  just  opened,  it  usually 
contains  a  great  number  of  particles  of  mucus,  some  large,  some  small. 
If  one  collects  the  liquor  in  a  bottle  and  allows  it  to  stand  over  night 
it  will  be  found  to  have  separated  into  two  distinct  layers,  a  heavy, 
thick,  viscous  layer  on  the  bottom  and  a  clear,  more  limpid  layer  on 
the  top.  The  bottom  layer  is  the  mucus  which  has  precipitated  out. 
Standard  Methods  of  Water  Analysis  requires  a  water  sample  to  be 
shaken  twenty-five  times  before  the  analysis  commences,  in  order  to 
break  up  any  clumps  of  bacteria.  The  second  Progress  Report  of  the 
Committee  on  Standard  Methods  of  Shellfish  Examination^  recom- 
mends that  "bacterial  counts  shall  be  made  of  a  composite  sample 
of  each  lot  obtained  by  mixing  the  shell  liquor  of  five  oysters.  Agar 
shall  be  used  for  the  culture  medium  and  in  general  the  procedure 
shall  be  in  accordance  with  the  method  recommended  by  the  com- 
mittee on  Standard  Methods  of  Water  Analysis  of  the  American 
Public  Health  Association. "  It  can  be  inferred  from  the  last  sentence 
of  the  quotation  that  it  includes  shaking  the  sample.  In  draining 
the  liquid  from  the  oyster  the  water  runs  out  of  the  shell  not  at  a 
single  point,  but  over  a  considerable  part  of  the  edge  of  the  shell. 

iJour.  Am.  Pub.  Health,  II,  1912,  34. 


24  BACTERIOLOGY  OF  THE  OYSTER. 

For  this  reason  the  mouth  of  the  ordinary  water  sample  bottle  is  not 
large  enough  to  collect  all  the  juice  and  so  in  most  laboratories  a 
sterile  petri  dish  is  used  for  the  purpose.  This  would  preclude  the 
possibility  of  shaking.  Now  if  shaking  of  a  water  sample,  which 
to  the  eye  is  perfectly  clear,  is  advisable  to  break  up  the  clumps  of 
bacteria  and  give  a  more  even  distribution  of  bacteria,  what  can  be 
said  of  the  juice  of  the  oyster  which  has  a  decided  milky  appearance 
and  which  usually  contains  strings  and  flakes  of  mucus  large  enough  to 
be  seen  several  feet  away?  If  one  plates  a  cubic  centimeter  of  this 
mixture  without  shaking,  the  flakes  will  appear  in  the  solid  medium 
as  irregular,  opaque  particles.  The  probabilities  are  from  the 
writer's  experience  that  the  flakes  of  mucus  carry  a  large  number  of 
bacteria  and  we  have  a  large  confluent  mass  of  colonies  developing 
around  each  mucus  flake.  Even  if  flakes  are  not  present,  laree 
confluent  masses  of  colonies  from  the  size  of  a  penny  to  the  size  of  a 
quarter  develop,  which  render  counting  impossible.  Usually,  how- 
ever, only  bile  tubes  are  used  for  the  presumptive  test  for  B.  coli  and 
no  plates  made  so  that  this  clumping  is  not  noticeable  except  where 
bile  tubes  do  not  duplicate  or  where  one  gets  a  positive  test  in  the 
1-lOth  c.c.  or  1-lOOth  c.c.  dilution  and  not  in  the  1  c.c.  or  a  positive 
presumptive  test  in  the  1-lOOth  c.c.  dilution  and  not  in  the  1  c.c. 
or  1-lOth  c.c.  dilution.  In  a  study  of  about  2,000  tubes  in  the  pre- 
sumptive test  the  writer  found  that  they  duplicated  only  about  two- 
thirds  of  the  time  and  that  in  one  set  one  might  get  a  positive  presump- 
tive test  in  the  1-lOOth  c.c.  dilution  and  in  the  duplicate  set  only  in  the 
1  c.c.  dilution  or  not  at  all. 

Aside  from  the  part  played  by  the  mucus  in  oyster  juice  the  part 
played  by  the  mucus  left  upon  the  body  of  the  oyster  is,  generally 
speaking,  much  more  important.  Often  much  more  mucus  is  left 
upon  the  body  of  the  oyster  than  is  found  in  the  oyster  juice.  As  the 
mucus  is  the  part  which  catches  the  bacteria  and  holds  them,  it  follows 
that  often  more  bacteria  are  left  upon  the  oyster's  body  than  are 
found  in  the  oyster  juice.  Hence,  it  follows  that,  if  we  only  examine 
the  juice  of  the  oyster  we  are  only  finding  a  fraction  of  the  bacteria 
really  present  in  the  oyster.  These  facts  will  be  brought  out  more 
clearly  when  the  experimental  work  upon  which  these  statements 
are  based,  is  taken  up. 

The  idea  of  comparing  the  number  of  bacteria  found  in  the  shell 
liquor  with  the  number  that  can  be  "washed"  from  the  body  of  the 


BACTERIOLOGY  OF  THE  OYSTER.  25 

oj^ster  is  not  new.  ^Houston  performed  a  series  of  experiments 
on  this  point  and  his  technique  and  results  are  given  in  some 
detail. 

EXPERIMENT  "A/' 

September  9th»  1903. 

"  The  oysters  utiHzed  for  this  experiment  were  gathered  in  the  Helford 
Kiver,  at  low  tide,  on  September  8th.  They  were  cleansed  in  the 
manner  described  elsewhere,  before  being  opened  with  a  sterile  knife. 
Each  oyster  was  carefully  detached  from  the  two  valves  of  its  shell, 
with  as  little  injury  as  possible,  and  washed  in  the  manner  about  to 
be  described. 

"A  sterilized  funnel  was  placed  in  a  sterile,  1,000  c.c.  measuring 
cj-linder  as  shown  in  the  accompanying  figure.  The  liquor  in  the 
oyster  shell  was  poured  into  the  cylinder  before  the  oyster  was 
completely  detached,  and  then  the  oyster  was  removed  from  the  shell 
with  sterile  forceps,  held  over  the  funnel,  well  washed  with  sterile 
water,  and  allowed  to  rest  in  the  funnel.  Ten  oysters  were  treated 
severally  in  this  manner,  and  then  allowed  to  drain  in  the  funnel. 

I.    LIQUOR. 

"  The  total  amount  of  sterile  water  employed  for  washing 

purposes  was 810  c.c. 

The  total  volume  of  liquid  (oyster  liquor  and  ''washings") 

in  the  measuring  cylinder  was 840  c.c. 

Therefore  the  volume  of  oyster  liquor  for  10  oysters  was 30  c.c. 

or  3  c.c.  liquor  per  oyster. 

"  The  funnel  containing  the  oysters  was  then  lifted  into  a  second 
sterile  cylinder,  and  sterile  water  was  poured  into  the  first  cylinder 
up  to  the  1,000  c.c.  mark. 

"  The  cultures  were  then  carried  out  in  the  ordinary  way  described 
elsewhere. 

Restjits  of  the  Examination  of  the  Liquor. 

"  Coli-like  microbes  were  isolated  in  pure  culture  from  1  c.c.  and  0.1 
c.c.  of  the  htre  of  mixed  oyster  liquor  and  sterile  water. 

^Loc.  cit. 

4 


26  BACTERIOLOGY  OF  THE  OYSTER. 

This  result  indicates  that  the  litre  consisting  of  oyster  liquor 
+  sterile  water  contained  coli-like  (apart  from  slow  liquefaction  of 
gelatine)  microbes  in  amount  corresponding  to  about  10  per  c.c. 
The  whole  litre  could  thus  be  considered  to  contain  about  10,000  coli- 
like  microbes  derived  from  30  c.c.  of  oyster  liquor. 

Hence,  if  10  oysters  yield  30  c.c.  of  liquor  containing  10,000  coli- 
like  microbes,  taking  the  average  liquid  contents  of  each  oyster  as 
3  c.c,  this  works  out  at  1,000  coli-like  microbes  in  the  hquid  contents 
of  each  oyster,  or  about  330  coli-like  microbes  per  c.c.  of  oyster  liquor. 

II.    OYSTERS. 

"  The  oysters  were  one  by  one  removed  from  the  funnel,  cut  up  with 
sterile  scissors,  and  placed  in  the  second  sterile  cylinder.  A  known 
quantity  of  sterile  water  (100  c.c.)  was  then  added,  the  total  volume 
read  off,  and  hence  after  deducting  100  c.c.  the  volume  of  the  oysters 
was  obtained.  It  was  found  to  be  90  c.c.  Sterile  water  was  then 
added  to  the  cylinder  until  the  volume  of  the  liquid  was  equal  to 
1,000  c.c. 

"  The  cultures  were  then  carried  out  in  the  ordinary  way  described 
elsewhere. 

Results  of  the  Examinations  of  the  Oysters*  Bodies. 

'  Coli-like  microbes  were  respectively  isolated  from  10  c.c.  and  1  c.c. 
of  the  litre  consisting  of  a  mixture  of  washed  oyster  bodies  and  sterile 
water. 

"  The  litre  consisting  of  sterile  water-f  macerated  oysters  might  be 
considered  to  contain  about  1 ,000  coli-like  microbes  derived  from  the 
bodies  of  10  oysters.  Therefore,  each  oyster  body  (deprived  as  far 
as  possible  of  its  natural  liquor)  would  contain  coli-like  microbes 
corresponding  in  number  to  about  100. 

"  The  total  volume  of  oyster  bodies  being  90  c.c,  the  volume  of  each 
of  the  10  oysters  averaged  9  c.c;  each  9  c.c.  of  oyster  body  could  be 
considered  to  contain  100  coli-like  microbes,  or,  roughly  speaking, 
11  coli-like  microbes  per  c.c  of  body  bulk.  The  contrast  is  very 
striking  when  this  number  is  compared  with  that  of  330  coli-like 
microbes  per  c.c.  of  oyster  liquor,  i.  e.,  volume  for  volume  the  oyster 
liquor  contains  about  30  times  as  many  coli-like  microbes  as  the  oyster 
body. 


BACTERIOLOGY  OF  THE  OYSTER. 


27 


"  But  the  liquid  contents  of  the  oyster's  stomach  are  certainly  much 
less  than  1  c.c,  probably  about  0.1  c.c.  It  is  probably  that  the  coli- 
like  microbes  isolated  from  the  macerated  oyster  bodies  in  the  fore- 
going experiment  were  totallj^,  or  in  great  part,  derived  from  the 
contents  of  the  stomach  and  intestinal  tract.  In  fact,  it  is  conceivable 
that  the  100  coli-like  microbes,  which  each  washed  oyster  was  found 
to  contain,  were  all,  or  to  a  great  extent,  derived  from  the  stomach 
juice  which,  for  comparative  purposes,  may  be  assumed  to  be  about 
0.1  c.c.  But  if  the  body  volume  of  each  oyster  be  taken  as  9  c.c,  the 
volume  of  the  stomach  contents  on  the  above  assumption  is  only  about 
one-ninetieth  of  the  total  bulk. 

"  This  view  alters  considerably  the  complexion  of  affairs.  For  the 
ratio  between  the  number,  per  unit  of  volume,  of  coli-like  microbes 
present,  respectively,  in  the  oyster  liquor  and  stomach  juice,  would 
then  be  33:100.  In  other  words,  acting  on  this  assumption  the  coli- 
like  microbes  were  three  times  more  numerous  per  unit  of  volume  in 
the  stomach  or  intestinal  juice  then  in  the  oyster  liquor. 


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*0n  the  assumption  that  all  the  coli-Uke  microbes  obtained  from  the  macerated  bodies  of  the 
oysters  were  derived  from  the  stomach  juice  (taking  the  volume  of  the  stomach  juice  as  O.lc.c. 
and  the  volume  of  the  oyster  apart  from  its  liquor  as  10c. c.)." 


28  BACTERIOLOGY  OF  THE  OYSTER. 

From  these  experiments  and  the  conclusions  drawn  it  is  clear  that 
Dr.  Houston  thought  that  all  the  bacteria  on  the  outside  of  the  oysters 
were  washed  off  with  the  sterile  water  used  to  wash  the  bodies  of  the 
oysters  and  that  all  the  organisms  found  in  the  minced  oysters  came 
from  the  stomach.  Whether  we  can  accept  Dr.  Houston's  supposition 
or  not  will  be  discussed  later  under  the  writer's  own  experiments  in 
this  connection. 

Stiles  in  the  bulletin  referred  to  above  made  some  analyses  showing 
the  relative  numbers  of  bacteria  in  the  shell  liquor  and  meat  of 
oysters.  He  concludes:  "The  results  show  that  the  oyster  liquor  in 
these  samples  contained  more  than  seven  times  as  many  organisms  per 
given  volume  as  did  the  minced  meat  and  body  contents  of  the  same 
oysters.  The  results  further  show  that  the  liquor  contained  eight 
times  as  many  B.  coli  per  cubid  centimeter  as  the  minced  meat." 

Stiles  does  not  give  his  method  of  determining  the  number  of  bacteria 
in  the  minced  body  of  the  oyster.  It  may  well  be  that  his  results 
actually  do  show  the  relative  numbers  of  bacteria  in  the  two  parts  of 
the  oyster.  His  experiments  included  the  results  of  only  fifteen 
analyses,  and  the  results  uniformally  show  a  greater  number  of 
bacteria  in  the  shell  liquor  than  in  the  minced  body  meat.  In  the 
light  of  the  writer's  results  of  similar  analyses,  however,  we  are  led 
to  believe  that  the  method  of  analysis  is  not  adequate  to  demonstrate 
the  relative  number  of  bacteria  in  the  two  parts  of  the  oyster.  It  is 
conceded  by  all  that  the  tissues  of  the  oyster  are  sterile.  It  is  only 
the  outside  of  the  body  and  the  alimentary  tract  which  normally 
harbor  bacteria.  It  is  easy  to  understand  how  so  much  minced 
tissue  will  interfere  with  accurate  results.  Secondly  no  mention  is 
made  of  how  the  bacteria  were  separated  from  the  minced  meat.  An 
immense  amount  of  shaking  would  be  necessary  to  make  an  even 
suspension  of  bacteria  if  one  tried  to  wash  them  from  the  minced 
particles  of  the  oyster  meat.  The  bacteria  are  attached  to  the  body 
of  the  oyster  by  the  mucus  which  is  not  easily  removed.  Even  though 
the  minced  oysters  were  shaken  vigorously  in  water  or  salt  solution, 
the  particles  would  quickly  settle  out  and  being  more  or  less  entangled 
in  the  mucus  a  coagulum  would  be  formed  which  setthng  out  rapidly 
would  take  a  great  many  if  not  most  of  the  bacteria  out  of  suspension. 
This  is  purely  suppositional  since  the  method  of  analysis  is  not  given, 
but  this  is  a  perfectly  logical  method  of  procedure  and  a  very  probable 
explanation  of  the  results.  The  temperature  of  the  water  from  which 
the  oysters  were  taken  is  not  given.     In  the  writer's  opinion  this  is 


BACTERIOLOGY  OF  THE  OYSTER.  29 

an  important  matter,  for  the  temperature  of  the  water  will  influence 
the  metabohsm  of  the  mucus  secreting  cells  and  will  determine  the 
amount  of  mucus  present  on  the  body  of  the  oyster.  This  matter  will 
be  discussed  further  in  another  connection. 

When  the  writer  began  his  experiments,  he  did  not  know  of  Hous- 
ton's work  and  so  the  experiments  were  not  carried  out  in  exactly 
the  same  manner,  but,  nevertheless,  the  experiments  throw  consider- 
able light  on  the  work  just  cited.  The  idea  that  the  mucus  of  the 
oyster  played  a  part  as  yet  unappreciated  led  the  writer  to  perform 
the  following  series  of  experiments. 

Experiment  I. 

September  29,  1913,  ten  oysters  were  t'aken  to  the  laboratory  and 
analyzed  as  follows:  The  oysters  were  opened  according  to 
"Standard  Methods"  and  the  hquor  drained  into  a  small  bottle 
graduated  in  two  cubic  centimeter  divisions.  The  oysters  were 
allowed  to  drain  until  a  drop  would  not  come  away  at  least  every  five 
seconds.  The  amount  of  liquor  was  then  read  off  and  an  equal 
volume  of  sterile  salt  solution  added  and  the  whole  shaken  vigorously 
one  hundred  times.  The  body  of  the  oyster  was  removed  from  the 
shell  and  placed  in  a  sterile  jar  and  a  quantity  of  sterile  salt  solution 
added  equal  to  the  volume  of  the  shell  liquor.  The  jars  were  covered 
and  allowed  to  stand  for  a  short  time  while  the  oyster  juice  was  being 
inoculated  into  plates  and  bile  tubes.  The  jars  containing  salt 
solution  and  oyster  meat  were  then  stirred  vigorously  with  a  sterile 
pipette  and  an  attempt  made  to  remove  with  the  pipette  as  much 
mucus  as  possible  from  the  body  of  the  oyster.  Then  one  cubic 
centimeter  of  the  solution  and  dilutions  thereof  were  inoculated  into 
plain  agar  plates  and  lactose-peptone-bile  in  the  same  manner  as  in 
the  case  of  oyster  juice.  A  careful  record  was  kept  of  the  number  of 
cubic  centimeters  of  juice  obtained  from  each  oyster  and  the  amount 
of  salt  solution  used  in  washing  each  oyster  in  order  to  make  a  com- 
parison of  the  bacterial  content  of  all  the  shell  liquor  with  the  total 
number  of  bacteria  washed  from  the  oyster.  This  would  show  which 
part  contained  the  greater  number  of  bacteria. 

Experiment  IL 

The  above  experiment  was  repeated  on  oysters  obtained  October  7, 
1913.     The  total  number  of  bacteria  found  in  the  shell  liquor  and  the 


30 


BACTERIOLOGY   OF    THE    OYSTER. 


washings  from  the  bodies  of  the  oysters  in  each  of  the  two  experiments 
is  sho-^Ti  in  the  following  table : 


Table  Showing  the  Total  Number  of  Bacteria  in  the  Shell  Liquor  of  each  Sarnple 
and  the  Total  Number  Washed  from  the  Bodies  of  the  Oysters  Without  Shaking. 


Sept.  29.     Shell  Liquor j  330,000 

"Washings" 48,000 

Oct.      7.     Shell  Liquor !  480,000 

"Washings" i  50,000 


7,400 

1,700 

5,900 

850 


The  detailed  results  are  sho^\^l  in  the  two  follo^^^ng  tables: 


1 
o 

o 
d 

1 
1 

•o 

6 
d 

20°C  Count. 

B.  co!i  Count. 

Score. 

Date. 

Number  of 
Bacteria 
in  Shell 
Liquor. 

Number  of 
Bacteria 
Washed 

from 
Oyster. 

Number 

in 

Shell 

Liquor. 

Number 
Washed 

from 
Oyster. 

Based 

on 
Shell 
Liqour. 

Based 
on  Shell 

Liquor 

and 

Washings 

Sept.  29,  1913. 

1 

2 
3 
4 
5 
6 
7 
8 
9 
10 

8 
4 
3 

10 
11 
10 
9 
13 
5 
10 

27,000 
4,000 
2,900 
51,000 
13,000 
14,000 
76,000 
14,000 
75,000 
58,000 

1,900 
1,600 
2,400 
2,400 
5,500 
4,800 
14,000 
4,700 
6,000 
4,600 

1,600 
80 

60 

200 

220 

200 

1,800 

260 

1,000 

2,000 

800 

400 

30 

10 

11 

100 
90 

130 
50 

100 

200 
20 
20 
20 
20 
20 

200 
20 

200 

200 

300 
120 

30 

21 

21 

30 

210 

30 

210 

210 

Totals.  .  .  . 

83     334,900 

i 

47,900 

7,420 

1,721 

920 

1,182 

BACTERIOLOGY   OF   THE    OYSTER. 


31 


i 

20°C  Count. 

B.  coli  Count. 

Score. 

Date. 

s 

3 

B 
? 

Number  of 

Number  of 

Number 

Number 

Based 

Based 
on  Shell 

S 

o 

Bacteria 

Wished 

in 

Washed 

on 

Liquor 

in  Shell 

shell 

from 

Shell 

and 

o 

d 

Liquor. 

Oyster. 

Liquor. 

Oyster. 

Liquor. 

Wash- 
ings. 

2 

o 

October    7,  1913  . 

1 

10 

40,000 

1,000 

200 

100 

20 

30 

' 

' 

2 

20 

20,000 

3,700 

2,000 

10 

100 

101 

' 

3 

12 

54,000 

20,000 

240 

10 

20 

21 

' 

4 

10 

70,000 

300 

20 

10 

2 

3 

' 

5 

18 

13,500 

1,200 

18 

100 

1 

61 

' 

' 

6 

14 

126,000 

12,000 

2,800 

200 

200 

214 

' 

' 

7 

IS 

73,800 

5,000 

180 

200 

10 

21 

' 

' 

8 

10 

12,000 

3,200 

200 

200 

20 

20 

A 

' 

9 

no 

12 

72,100 

3,400 

240 

0 

20 

20 

Totnls. 

481,300 

49,800 

5,898 

830 

393 

511 

*Not  examined. 


It  will  be  noticed  that  in  the  last  two  columns  of  the  table  is  given 
the  score  based  upon  the  shell  liquor  alone  and  upon  the  shell  liquor 
and  the  "washings"  from  the  oyster  combined.  The  method  of 
scoring  is  based  upon  the  same  principal  as  the  method  of  scoring 
recommended  by  "Standard  Methods,"  but  it  works  out  a  little 
differently  for  the  method  of  analysis  followed  by  the  writer  is  not 
strictly  in  accordance  with  "Standard  Methods."  In  the  latter 
method  1  c.c,  1-10  c.c.  and  1-100  c.c.  quantities  of  the  shell  liquor 
are  inoculated  into  lactose-peptone-bile.  If  the  presumptive  test 
shows  B.  coli  in  1  c.c.  dilution  and  not  in  the  1-10  c.c.  and  the  1-100 
c.c.  then  the  score  of  this  oyster  is  one.  If  it  shows  B.  coli  in  1-10  c.c. 
and  not  in  1-100  c.c,  the  score  is  ten;  if  in  1-100  c.c.  the  score  is  100. 
In  other  words,  the  score  of  the  oyster  equals  the  number  of  B.  coli 
found  in  one  cubic  centimeter  of  the  shell  liquor.  In  the  writer's 
experiments  the  shell  liquor  was  carefully  measured  and  diluted  wath 
an  equal  volume  of  one  per  cent.  NaCl  solution.  One  cubic  centi- 
meter of  this  mixture  was  used  to  make  the  various  dilutions.  The 
result  is  that  the  various  dilutions  contained  3^  c.c,  %o  c.c  and 


32  BACTERIOLOGY  OF  THE  OYSTER. 

^/^oo  c.c.  of  the  shell  liquor.  Gas  appearing  in  these  respective 
dilutions  would  indicate  two,  twenty  and  two  hundred  B.  coli  per  cubic 
centimeter  instead  of  one,  ten  and  one  hundred  as  in  the  procedure 
of  "  Standard  Methods. " 

The  last  column  gives  the  combined  score,  in  other  words,  the  score 
based  upon  the  number  of  B.  coli  in  both  the  shell  liquor  and  in  the 
washings.  The  number  of  B.  coli  in  each  is  added  together  and 
divided  by  the  number  of  cubic  centimeters  of  shell  liquor.  This 
method  makes  no  allowance  for  the  amount  of  mucus  present  on  the 
body  of  the  oyster.  This  quantity  would  not  exceed  one  cubic 
centimeter  on  the  average,  for  many  of  the  oysters  were  small.  It  is 
more  convenient  and  just  as  accurate  for  comparative  purposes  to 
ignore  this  quantity,  while  it  is  much  more  convenient  in  dividing  the 
total  number  of  B.  coli  found  in  the  oyster  by  the  quantity  of  shell 
liquor.  It  avoids  fractions  much  more  often  than  would  be  the  case 
if  we  added  one  to  the  number  of  cubic  centimeters  of  shell  liquor. 
Occasionally,  however,  in  the  combined  score,  the  quotient  is  not  an 
even  number  and  so  the  score  is  made  the  whole  number  next  above 
or  below  depending  whether  the  fraction  was  less  than  or  more  than 
one-half.  Thus  if  the  score  came  20.4  it  would  be  called  20;  if  the 
fraciton  were  .5  or  more  it  would  be  called  21. 

It  would  seem  from  these  experiments  that  there  is  no  question  that 
the  shell  liquor  contains  many  more  bacteria  than  are  left  on  the  body 
of  the  oyster  and  that  in  analysis  we  could  ignore  entirely  the  bacteria 
left  on  the  body  of  the  oyster. 

These  results  did  not  equal  the  writer's  expectation  and  it  was 
thought  that  perhaps  the  treatment  of  the  oyster's  body  was  not  suf- 
ficient to  remove  all  the  mucus  and  bacteria  present.  Accordingly  the 
following  method  of  analysis  was  adopted  for  the  subsequent  experi- 
ments :  The  oyster  liquor  was  collected  and  diluted  in  the  same  manner 
as  before.  It  was  shaken  vigorously  one  hundred  times  before  inocu- 
lating into  agar  and  bile.  The  body  of  the  oyster  after  draining  was 
transferred  to  a  sterile  large  mouthed,  glass  stoppered  bottle  and 
covered  with  twenty  cubic  centimeters  of  one  per  cent.  NaCl  solution. 
The  oyster  and  salt  solution  were  shaken  fairly  vigorously  one  hundred 
times  and  the  solution  of  salt  and  mucus  was  removed  by  the  pipette 
or  poured  into  a  smaller  glass  bottle  and  again  shaken  vigorously 
one  hundred  times.  This  mixture  was  then  inoculated  into  the  bile 
tubes  and  the  agar  plates.     At  first  one  per  cent,  sodium  carbonate 


BACTERIOLOGY  OF  THE  OYSTER.  33 

solution  was  used  with  the  hope  that  it  would  cut  the  mucus  more 
readily,  but  later  the  salt  solution  was  found  just  as  effective.  The 
shaking  appeared  to  be  the  important  feature. 

It  was  found  that  a  great  deal  of  shaking  was  necessary  to  break 
up  the  clumps  of  bacteria  and  separate  them  from  the  mucus.  If 
not  thoroughly  shaken  the  resulting  plates  would  be  found  to  contain 
large  areas  of  confluent  colonies  which  rendered  counting  impossible. 
Every  bit  of  mucus  would  be  found  to  be  a  nucleus  around  which 
would  be  a  large  confluent  ring  of  colonies.  After  a  thorough  shaking, 
however,  the  flakes  of  mucus  would  in  nearly  all  cases  remain  sterile 
and  the  bacteria  would  be  found  in  well  separated  colonies  evenly 
distributed  in  the  medium. 


34 


BACTERIOLOGY   OF   THE    OYSTER. 


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BACTERIOLOGY  OF  THE  OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY  OF  THE  OYSTER.  43 

In  comparing  the  total  number  of  bacteria  in  the  shell  liquor  of  all 
the  oysters  in  each  of  the  experiments  with  the  total  number  washed 
from  the  bodies  of  these  oysters  it  is  seen  that  the  total  number  of 
bacteria  in  the  shell  liquor  of  all  the  oysters  was  greater  than  the 
number  washed  from  the  bodies  of  the  oysters.  In  the  first  experi- 
ment the  numbers  are  nearly  equal,  but  in  the  subsequent  experiments 
there  is  a  great  difference.  If  we  consider  the  individual  oysters  in 
all  the  experiments,  we  find  that  in  only  ten  of  the  oysters  out  of 
seventy-seven  was  there  a  greater  number  of  bacteria  washed  from 
the  body  than  was  found  in  the  shell  liquor  of  the  corresponding 
oyster.  In  one  instance  the  numbers  were  equal.  In  the  remaining 
sixty-six  oysters  there  were  more  bacteria  in  the  shell  liquor  that  were 
washed  from  the  bodies  of  the  oysters.  The  37°  C.  count  and  the 
"red  count"  were  made  on  only  seventeen  oysters  and  in  only  two 
instances  did  the  number  of  bacteria  washed  from  the  bodies  of  the 
oysters  exceed  the  number  found  In  the  shell  liquor,  while  the  total 
number  from  all  the  oysters  of  the  two  experiments  showed  that  there 
were  on  the  average  a  great  many  more  in  the  shell  liquor  than  were 
washed  from  the  bodies  of  the  oysters. 

When  we  consider  the  number  of  B.  coli  found  in  the  shell  liquor 
and  the  number  washed  from  the  body  of  the  same  oyster  we  find  the 
relative  numbers  quite  different.  It  will  be  seen  in  six  out  of  the  eight 
experiments  the  total  number  of  B.  coli  washed  from  the  bodies  of  all 
the  oysters  of  the  experiment  exceeded  the  total  number  in  the  shell 
liquor.  In  the  first  two  experiments  the  difference  is  especially 
marked.  If  we  consider  the  individual  oysters  we  find  that  in  thirty- 
three  instances  there  were  more  B.  coli  on  the  body  of  the  oyster  than 
were  in  the  shell  liquor;  in  thirty  oysters  the  number  in  the  shell 
liquor  exceeded  the  numl^er  washed  from  the  body;  in  fourteen 
instances  the  numbers  were  equal.  But  if  we  consider  the  total 
number  of  B.  coli  found  in  the  "washings"  with  the  total  number 
found  in  the  shell  liquor  of  all  the  oysters  examined  in  this  series  of 
experiments  we  find  there  were  on  the  average  more  B.  coli  in  the 
"  washings  "  than  there  were  in  the  shell  liquor. 

We  have  no  reason  at  present  to  suppose  that  B.  coli  should  be 
distributed  other  than  equally  among  the  other  bacteria  in  the  oyster, 
yet  there  seems  to  be  a  concentration  of  B.  coli  in  the  mucus  on  the 
outside  of  the  body  of  the  oyster.  The  amount  of  shell  liquor  in  the 
oysters  averaged  about  ten  cubic  centimeters.  If  we  consider  that 
there  was  left  upon  the  body  of  the  oyster  one  cubic  centimeter  of 


44 


BACTERIOLOGY    OF   THE    OYSTER. 


mucus,  we  find  that  there  were  volume  for  volume  more  than  ten 
times  as  many  B.  coli  on  the  body  of  the  oyster  as  there  were  in  the 
shell  liquor.  The  question  arises  at  once  as  to  whether  this  unequal 
distribution  of  B.  coli  among  the  other  bacteria  in  these  two  parts 
of  the  oyster  is  real  or  only  apparent.  It  may  be  due  to  the  difference 
in  methods  of  analysis.  With  our  present  knowledge  of  the  bacterio- 
logy of  the  oyster  the  writer  is  led  to  believe  that  this  relation  does 
not  actually  exist,  but  is  due  to  the  difference  in  methods  used  to 
determine  the  total  number  of  bacteria  and  the  number  of  B.  coli. 

Another  point  which  appears  interesting  to  the  writer  is  that  there 
is  apparently  a  direct  relation  between  the  temperature  of  the  water 
from  which  the  oysters  are  taken  and  the  relative  number  of  B.  coli 
found  in  the  shell  liquor  and  on  the  body  of  the  oyster.  It  will  be 
noticed  that  in  the  first  three  experiments  there  were  a  great  many 
more  B.  coli  on  the  body  of  the  oyster  than  in  the  shell  liquor,  but 
this  proportion  is  gradually  reduced  and  in  the  sixth  and  eighth  experi- 
ments there  were  more  B.  coli  in  the  shell  liquor  than  were  found  on 
the  bodies  of  the  oysters.  The  ratio  of  the  total  number  of  bacteria 
in  the  shell  liquor  to  the  total  number  washed  from  the  bodies  of  the 
oysters  in  each  sample  is  shown  in  the  following  table : 

Table  Arranged  According  to  Temperature  Showing  the  Approximate  Ratio  of  the 

Total  Number  of  Bacteria  in  the  Shell  Liquor  to  the  Number  in  the  Wash- 

ings  from  the  Bodies  of  the  Oysters  in  each  Sample. 


Temperature. 

Date. 

20°C. 
Count. 

37°C. 
Count. 

"Red" 
Count. 

B.  ooli 
Count. 

16°      C 

Oct.  13 
"  27 
"  23 
"      31 

Nov.  3 
"        8 

Dec.  6 
"      19 

1.3:1 
2:1 
4.2:1 
2:1 
5.2:1 
7.5:1 
6.5:1 
3.7:1 

1:3 

16°      C. 

1:18 

13  5°  C.  . 

1:2.8 

13°      C 

1:1.3 

12°      C 

1.3:1 

12°      C 

2:1 

8°      C. 

6:1 
10:1 

7:1 

4.7:1 

1:1  1 

6°      C.  .    . 

3:1 

A  long  series  of  experiments  necessitating  the  examination  of  a 
great  number  of  oysters  and  extending  over  a  whole  year  would  be 
required  to  establish  this  relationship.  However,  this  supposition 
is  not  so  different  from  what  we  might  expect  when  we  consider  the 


BACTERIOLOGY  OF  THE  OYSTER.  45 

biology  of  the  oyster.  The  optimum  temperature  for  the  growth  of 
the  oyster  is,  probably  between  20°  C.  and  25°  C.  At  this  temperature 
the  cells  of  the  oyster  are  most  active.  The  mucus  sells  will  secrete 
a  larger  amount  of  mucus  than  at  decidedly  lower  temperatures. 
The  more  mucus  secreted  the  more  will  remain  clinging  to  the  body 
of  the  oyster.  Generally  speaking  the  greater  the  amount  of  mucus 
the  greater  the  number  of  bacteria  we  would  expect  to  find  in  the 
mucus  on  the  outside  of  the  body.  As  the  temperature  of  the  water 
lowers,  the  metabolic  processes  of  the  oysters  are  correspondingly 
slowed  and  a  smaller  amount  of  mucus  and  for  this  reason  fewer 
bacteria  will  be  found  on  the  body  of  the  oyster.  For  this  reason  it 
seems  fair  to  assume  that  the  apparent  relation  between  the  tempera- 
ture of  the  water  and  the  proportion  of  B.  coli  on  the  outside  of  the 
oyster  and  the  shell  liquor  is  real  and  not  accidental. 

These  two  sets  of  experiments  throw  light  on  the  findings  of  Houston 
cited  above.  It  is  easily  seen  that  simply  pouring  water  over  the 
body  of  the  oyster  is  not  sufficient  to  remove  all  the  bacteria.  The 
experiments  of  the  writer  on  the  comparison  of  the  bacterial  content 
of  the  stomach  and  shell  liquor  shows  that  per  unit  volume  the  shell 
liquor  contains  on  the  average  over  twenty  times  as  many  bacteria 
as  the  stomach  juices.  Evidence  from  all  sides  shows  that  Houston's 
assumption  that  all  the  bacteria  were  washed  from  the  body  of  the 
oyster  by  simply  pouring  water  over  the  oyster  and  further  that  the 
bacteria  found  in  the  minced  meat  of  the  oysters  so  treated  came 
entirely  from  the  stomach  are  not  in  accordance  with  the  facts. 

These  experiments  show  the  necessity  of  examining  not  only  the 
shell  liquor,  but  also  the  mucus  on  the  outside  of  the  body  of  the  oyster. 
This  is  especially  true  during  the  warmer  months.  At  this  time 
there  are  on  the  average  many  more  B.  coli  on  the  body  of  the  oyster 
than  is  contained  in  the  shell  liquor.  It  is  perfectly  legitimate  to 
consider  the  mucus  on  the  body  of  the  oyster  as  part  of  the  oyster 
juice.  If  we  so  consider  the  mucus,  it  makes  a  very  decided  difference 
in  the  score  of  the  oyster.  In  one  instance  the  combined  score  of  one 
oyster  was  ninety-six  times  the  score  based  upon  the  shell  liquor  alone. 
The  combined  score  is  never  less  and  often  many  times  more  than  the 
score  based  upon  the  shell  liquor.  If  there  were  any  constant 
relation  between  the  B.  coli  content  of  the  shell  liquor  and  the  mucus 
removed  from  the  body  of  the  oyster,  the  examination  of  the  shell 
liquor  alone  would  be  sufficient.  But  as  no  such  relation  exists  the 
necessity  of  examining  both  the  shell  liquor  and  the  mucus  is  at  once 
apparent. 


46  BACTERIOLOGY  OF  THE  OYSTER. 

COMPARISON     OF    THE    BACTERIAL    CONTENT    OF    THE 
STOMACH  AND  OF  THE  SHELL  LIQUOR  OF  OYSTERS. 

Houston  in  his  report  to  the  local  Government  Board,  1904,  makes 
the  following  statement:  ''The  experiments  detailed  elsewhere  seem 
to  indicate  that  per  unit  of  volume  the  gastric  juice  of  the  oyster  is 
more  impure  bacteriologically  than  the  oyster  liquor."  The  experi- 
ments upon  which  this  statement  is  based  are  taken  up  in  some  detail 
under  "Bacteriology  of  the  Shell  Liquor  and  'Washings'  from  the 
Body  of  the  Oyster,"  and  so  it  is  not  necessary  to  take  up  these 
experiments  in  this  connection.  The  -writer  has  shown  that  these 
experiments  and  the  conclusions  drawn  are  not  based  upon  sound 
assumptions  and  so  these  results  are  not  to  be  relied  upon. 

Clark, ^  in  a  long  series  of  experiments  has  shown  that  in  both 
clams  and  oysters  the  shell  liquor  is  much  more  likely  to  yield  B.  coli 
or  sewage  streptococci  than  either  the  stomach,  intestine  or  rectum. 

Both  of  these  workers  studied  the  B.  coli  content  of  the  different 
parts  of  the  oyster.  The  writer  could  not  obtain  any  badly  polluted 
oysters  at  the  time  of  year  during  which  the  experiments  were  con- 
ducted and  so  he  examined  the  shell  liquor  and  stomach  contents 
for  the  total  number  of  bacteria  which  each  part  contained.  It 
would  have  been  possible  to  infect  oysters  artificially  with  the  colon 
bacillus,  but  it  is  not  certain  that  one  could  simulate  natural  conditions 
exactly  and  consequently  wrong  conclusions  might  be  draw^n.  We 
have  no  reason  to  suppose  that  B.  coli  are  distributed  other  than 
equally  among  the  other  bacteria  in  the  oyster  and  so  a  comparison 
of  the  total  quantity  per  unit  volume  ought  to  show  the  relative 
frequency  with  which  one  would  expect  to  find  any  particular  bacter- 
ium in  either  part  of  the  oyster. 

In  this  series  of  experiments  forty-one  oysters  were  used.  The 
method  of  examination  was  as  follows : — The  juice  of  each  oyster  was 
collected  in  a  small  glass-stoppered  bottle  which  was  calibrated  in  two 
cubic  centimeter  divisions.  The  amount  of  shell  liquor  was  read  off 
in  cubic  centimeters  and  diluted  with  an  equal  amount  of  one  per  cent, 
sodium  chloride  solution.  The  shell  liquor  and  the  sodium  chloride 
solution  were  shaken  vigorously  one  hundred  times  and  one  cubic 
centimeter  of  this  mixture  was  transferred  to  a  tube  containing  nine 
cubic   centimeters  of  one  per  cent,  sodium  chloride  solution  and 

^Report  State  Board  of  Health  of  Mass.  1905,  428. 


BACTERIOLOGY  OF  THE  OYSTER.  47 

one  cubic  centimeter  of  this  dilution  was  plated  in  agar.  Also  a 
cubic  centimeter  from  this  tube  was  transferred  to  another  tube  in 
nine  cubic  centimeters  of  salt  solution.  A  cubic  centimeter  of  this 
mixture  was  also  plated.  By  this  method  of  dilution  the  plates 
contained  respectfully  one  twentieth  and  one  two  hundredth  of  a  cubic 
centimeter  of  the  original  shell  liquor.  The  plates  were  made  in 
duplicate.  After  the  oyster  had  been  drained  of  its  liquor  the  fiat 
valve  was  removed  and  the  other  valve  containing  the  body  of  the 
oyster  was  set  on  the  edge  and  allowed  to  drain  for  several  minutes. 
The  excess  of  liquor  was  then  removed  with  a  piece  of  blotting  paper 
and  the  region  over  the  stomach  was  seared  with  a  hot  spatula  and  an 
incision  made  into  the  stomach  with  a  sterile  scalpel.  With  a  gradu- 
ated pipette  one-twentieth  of  a  cubic  centimeter  of  the  stomach 
contents  was  removed  and  plated  another  twentieth  of  a  cubic  centi- 
meter was  transferred  to  a  tube  containing  nine  cubic  centimeters 
of  salt  solution  and  1  cubic  centimeter  of  this  mixture  plated.  These 
plates  contained  respectfully  one-twentieth  and  one-two  hundredth 
of  a  cubic  centimeter.  The  plates  were  also  made  in  duplicate  and 
in  all  cases  the  average  of  the  two  plates  was  taken  as  the  count  for 
each  oyster.  The  counts  given  in  the  table  below  are  for  one-twentieth 
of  a  cubic  centimeter  of  the  oyster  juice  and  the  stomach  contents. 


48 


BACTERIOLOGY    OF   THE    OYSTER. 


Table  Comparing  the  Number  of  Bacteria  in  One-Twentieth  of  a  Cubic  Centimeter 

of  the  Shell  Liquor  with  the  Number  of  an  Equal  Quantity  of  the 

Stomach  Contents. 


No.  OF  Oyster. 

No.  ot  bacteria 

in  shell  liquor 

of  oyster. 

No.  of  bacteria 
in  stomach  con- 
tents of  oyster. 

1                       

20 

220 

8 

130 

16 

1 

50 

90 

20 

110 

3 

85 

12 

17 

430 

55 

1,000 

95 

500 

260 

340 

120 

340 

155 

1,000 

725 

38 

1,000 

1,000 

90 

19 

50,000 

10,000 

100,000 

15,000 

10,000 

24,000 

10,000 

50,000 

50,000 

1 

2               

5 

3                                                        

1 

4                                                

6 

5                                  

1 

6 .  . 

11 

3 

8                 

2 

9            

8 

10                                                        

13 

11                                                

9 

12                                    

6 

13                               

0 

14                     

4 

15 

3 

16                                                              

4 

17                                                          

1 

18                                                

1 

19                                            

2 

20                                    

1 

21                           

1 

22             

1 

23                                                                ■  ■  ■  • 

1 

24                                                             

0 

25                                                       

2 

26                                                 

1 

07 

6 

28 

2 

29                                                                .  .  .  . 

20 

30                                                            

3 

31                                                      

12 

32            .  .        .                      

14 

33 

470 

34 

280 

35                                                              

150 

36                                                        

10,000 

37 

10 

38 

30 

39 

40 

10 
2,500 

41 

25 

Totals 

304,351 

13,620 

BACTERIOLOGY    OF   THE    OYSTER.  49 

From  the  table  it  is  seen  that  in  only  two  oysters,  numbers  six  and 
eleven,  out  of  the  forty-one  examined,  was  the  number  of  bacteria 
per  unit  volume  greater  in  the  stomach  contents  than  in  the  shell 
liquor.  In  oyster,  twenty-eight  the  numbers  were  equal.  In  the 
remaining  thirty-eight  oysters  there  were  more  bacteria  per  unit 
volume  in  the  shell  liquor  than  in  the  stomach  contents.  In  these 
thirty-eight  oysters  the  ratio  per  unit  quantity  of  the  number  of 
bacteria  in  the  shell  liquor  to  the  number  in  the  contents  of  the 
stomach  varied  from  3  to  2  in  oyster  Nos.  37  to  2000  to  1  in  oyster 
No.  41.  The  ratio  of  the  total  numl^er  of  bacteria  per  unit  volume 
in  the  shell  liquor  of  the  forty-one  oysters  to  the  total  number  of 
bacteria  in  an  equal  quantity  of  the  stomach  contents  was  as  21.6  to  1. 
That  is,  a  comparison  of  the  average  number  of  bacteria  found  in  shell 
liquor  with  the  number  of  bacteria  in  the  stomach  contents  shows  that 
per  unit  quantity  there  were  more  than  twenty  times  as  many  bacteria 
in  the  shell  liquor  as  in  the  stomach  juice. 

LENGTH  OF  TIME  NECESSARY  FOR  BACTERIA  TO  PASS 
THROUGH  THE  INTESTINAL  TRACT  OF  THE  OYSTER. 

So  far  as  the  writer  is  aware  no  one  has  ever  made  any  determination 
of  the  rate  at  which  food  passess  through  the  alimentary  tract  of  the 
oyster.  While  it  is  difficult  to  determine  this  matter  directly,  it 
seemed  possible  to  inoculate  the  shell  liquor  of  oysters  with  some 
bacterium  not  found  in  oysters  and  trace  its  progress  through  the 
intestinal  canal.  B.  prodigiosus  was  chosen  because  of  its  ease  of 
identification  and  because  the  writer  has  never  found  it  in  oysters, 
and  so  far  as  he  is  aware  it  has  never  been  reported  as  occurring  in 
oysters. 

In  the  first  experiment  twelve  oysters  were  inoculated  by  sawing  off 
a  piece  of  the  lip  of  the  shell  and  inserting  a  loopful  of  a  culture  of  B. 
prodigiosus  into  the  branchial  chamber.  The  oysters  were  layed  very 
carefully  upon  cotton  tiioroughly  saturated  with  water  and  covered 
wdth  a  glass  dish  to  prevent  evaporation.  They  were  kept  at  the 
laboratory  temperature  which  is  about  20°C.  At  various  intervals, 
as  shown  in  the  tables,  three  oysters  were  removed  and  examined.  The 
examination  was  made  as  follows: — The  right  valve  of  the  oyster 
was  removed  and  the  gills  and  mantle  carefully  dissected  away.  The 
remaining  part  of  the  body  was  then  washed  for  several  minutes  in 
running  tap  water.     The  left  valve  containing  the  oyster  was  then 

7 


50 


BACTERIOLOGY   OF   THE    OYSTER. 


set  on  edge  and  allowed  to  drain  thoroughly.  The  surplus  water  was 
removed  with  filter  paper.  The  oyster  was  then  seared  with  a  hot 
spatula  over  the  stomach,  over  the  intestine,  where  it  bends  sharply 
upon  itself  on  the  ventral  side  and  on  the  rectum  just  above  the  anus. 
An  incision  was  then  made  into  these  three  parts  of  the  alimentary 
tract  with  sterile  scalpels  and  a  sterile  capillary  pipette  inserted  and  a 
portion  of  the  contents  removed  and  plated  upon  agar  which  was 
grown  at  room  temperature  for  two  days  and  examined  for  red  colo- 
nies. Control  samples  of  the  shell  liquor  were  plated  before  the  inocu- 
lation with  B.  prodigiosus  and  these  were  negative  in  all  cases.  In 
the  first  experiment  the  time  of  examination  after  inoculation  ranged 
from  thirteen  hours  to  twenty-seven  hours.  In  the  second  experiment 
the  time  varies  from  five  hours  to  seventy-four  hours. 


Table  Shoiving  Length  of  Time  at  which  B.  Prodigiosus  was  Isolated  from  Different 
Parts  of  the  Alimentary  Tract  after  the  Inoculation  of  the  Gill  Chamber. 


No.  OF  Oyster. 

Hours  after 
inooulation. 

Stomach. 

Intestine. 

Rectum. 

Experiment  I. 
1      

13 
13 
13 

18 
18 
18 
23 
23 
23 
27 
27 
27 

5 

5 

5 
22 
22 
22 
48 
48 
48 
74 
74 
74       ' 

+ 
+ 

0 
0 

+ 

0 
0 
•     0 
0 
0 
0 

+ 

0 
0 

+ 

0 
0 

+ 

0 
0 
0 
0 
0 
0 

0 
0 

+ 

0 
0 

+ 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

+ 

0 

0 

2    

0 

3 

0 

4 

0 

5 

0 

6                                   

+ 

7                             

0 

8          

0 

9    

+ 

10 

0 

11 

0 

12 

+ 

Experiment  11. 
1 

0 

2 

0 

3                                          .... 

0 

4                                

0 

5    

0 

6 

0 

7 

0 

8                                    

0 

9               

+ 

10 

11 

+ 
+ 

12 

0 

BACTERIOLOGY    OF    THE    OYSTER.  51 

From  these  tables  it  can  be  seen  that  the  first  appearance  of  the 
bacteria  in  the  intestine  was  thirteen  hours  after  inoculation  and  in  the 
rectum  five  hours  later.  We  would  expect  to  find  the  organisms  in 
the  stomach  within  a  very  short  time  after  inoculation  of  the  shell 
liquor.  Since  these  experiments  are  few  in  number  one  must  neces- 
sarily be  conservative  in  the  conclusion  drawn. 

THE  BACTERIAL  CONTENT  OF  OYSTERS  DURING  STORAGE. 

The  change  in  the  bacterial  content  of  oysters  during  storage  at  a 
temperature  at  which  they  are  kept  in  oyster  houses  and  during 
transportation  is  a  matter  of  very  great  importance  from  the  point 
of  view  of  the  public  health.  The  oyster  is  a  living  organism  capable 
of  maintaining  itself  for  a  long  period  when  removed  from  its  natural 
element.  It  is  possible  that  the  digestive  juices  or  the  phagocytic 
cells  of  the  oyster  might  materially  decrease  the  number  of  bacteria 
in  the  oyster.  On  the  other  hand,  even  if  the  digestive  secretions  and 
the  phagocytic  cells  were  bactericidal,  it  is  possible  that  the  rapid 
multiplication  of  the  bacteria  in  the  shell  liquor  might  be  sufficient 
to  maintain  or  increase  the  number  of  bacteria  in  the  oyster  as  a  whole. 
In  order  to  observe  the  change  in  the  bacterial  content  of  oysters 
during  storage  the  writer  carried  out  the  following  experiment : 

About  a  bushel  of  polluted  oysters  were  taken  from  the  Providence 
River  December  5,  1913.  and  put  into  storage  in  the  Laboratory  at  an 
average  temperature  of  10®C.  The  temperature  was  fairly  constant 
and  did  not  rise  above  11°C.,  although  for  a  short  time  during  a  period 
of  exceptionally  cold  weather  the  temperature  fell  to  8°C.,  but  it 
soon  rose  again  to  10°C.  The  oysters  were  put  into  storage  in  the 
bag  just  as  they  were  brought  to  the  laboratory.  No  attempt  was 
made  to  clean  them  in  any  way.  As  soon  as  they  arrived  a  sample 
of  ten  oysters  was  taken  from  the  bag  and  put  on  ice  and  examined 
the  following  day.  At  intervals  other  samples  of  ten  oysters  were 
removed  and  examined.  The  method  of  examination  was  the  same 
as  that  described  under  "The  Bacteriology  of  the  Shell  Liquors  and 
the  Washings  from  the  Bodies  of  the  Oysters."  In  all  except  two 
instances  a  20''C.  count,  a  37°C.  count,  a  "red"  count,  and  a  B.  coH 
count  were  made.  The  detailed  analysis  of  each  oyster  and  the 
bacterial  content  of  each  sample  as  a  whole  is  shown  in  the  following 
table: 


52 


BACTERIOLOGY    OF    THE    OYSTER. 


5^§ 


88 


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o  o 


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^  CO 


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o  o 

88 


o  o 
o  o 


88 

05    O 


88     88 


88 


o  o 

o  o 

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88     ^ 


o  o 
o  o 
o  o 


o  o 
o  o 
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BACTERIOLOGY    OF   THE    OYSTER. 


53 


1 

Based 

on  Shell 

Liquor 

and 

Washings. 

CO    (M    Tt<    O    (M    1-1    ■* 

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1 
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Number  of 
Bacteria 
Washed 

from 
Oyster. 

o  o  o  o  o  o  o 

5- 

Number  of 
Bacteria 
in  Shell 
Liquor. 

rf         T^               cf 

s 

a 
o 

Number  of 
Bacteria 
Washed 

from 
Oyster. 

2,800 

0 

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2,000 
500 
600 
200 

CO" 

16,000 
3,000 
5,600 
1,600 
5,400 
5,100 
2,400 

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Number  of 
Bacteria 
Washed 

from 
Oyster. 

gggSSsS 

0_  CO    -*^  C^T_  (M_  00    t- 

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67,000 
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3,700 
4,600 

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54 


BACTERIOLOGY   OF   THE    OYSTER. 


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BACTERIOLOGY   OF   THE    OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY   OF   THE    OYSTER. 


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62  BACTERIOLOGY  OF  THE  OYSTER. 

The  results  are  so  irregular  that  we  can  draw  no  very  specific 
conclusions.  It  appears  that  in  the  first  two  weeks  there  is  no  initial 
decrease  but  rather  a  steady  increase  in  the  total  number  of  bacteria 
present.  This  increase  is  also  apparent  in  the  ST^C.  count  and  the 
"red"  count.  On  January  27,  fifty-three  days  after  the  beginning 
of  the  experiment  there  was  a  remarkable  change  in  the  proportion 
of  bacteria  in  the  "washing"  as  compared  with  the  shell  liquor  in 
all  except  the  B.  coli  count.  The  detailed  analsyis  for  this  date 
shows  that  oyster  number  nine  is  responsible  for  this  marked  change. 
It  is  very  probably  that  this  oyster  had  died  and  decomposition  was 
taking  place. 

The  B.  coli  count  shows  a  decrease  on  the  fourth  day,  but  this 
decrease  is  not  particularly  marked  and  may  well  be  due  to  variations 
in  the  oysters  and  not  to  an  actual  decrease.  This  is  all  the  more 
likely  when  it  is  found  that  on  the  seventh  day  the  number  of  B.  coli 
is  approximately  the  same  as  on  the  first  day.  The  subsequent 
examinations  show  that  the  number  of  B.  coli  is  about  one-half  the 
initial  number  and  remains  fairly  constant  throughout  the  experi- 
ment. In  the  last  analysis  made,  eighty  days  after  the  beginning  of 
the  experiment,  all  the  bile  tubes  showing  gas  after  twenty-four  hours 
incubation  were  tested  for  B.  welchii  by  inoculating  a  cubic  centimeter 
of  the  bile  into  freshly  sterilized  milk  tubes  and  incubating  anaerobi- 
cally.  No  visible  change  took  place  in  the  milk  after  eighteen  hours 
incubation.  It  was  a  noticeable  fact  that  not  over  ten  per  cent,  of  the 
tubes  showing  gas  after  twenty-four  hours  incubation  had  one 
hundred  per  cent,  of  gas,  the  amount  said  to  be  characteristic  of 
B.  welchii.  Most  of  the  tubes  had  about  fifty  per  cent.  gas.  From 
these  facts  it  appears  that  the  fermentation  was  caused  by  some  mem- 
ber of  the  B.  coli  group  and  not  by  B.  welchii.  It  is  not  surprising 
to  find  that  B.  coli  should  live  eighty  days  in  oysters  under  such 
conditions  for  Clark^  has  shown  that  B.  coli  will  live  in  ten  per  cent, 
sewage  eighty-four  days. and  in  fifty  per  cent,  sewage  one  hundred  and 
sixty-six  days.  Unpublished  results  from  this  laboratory  show  that 
B.  coli  will  live  in  sea  water  for  one  hundred  and  eighty  days.  The 
writer  has  shown  in  the  experiments  on  the  hibernation  of  the  oyster 
that  B.  coli  will  live  in  oysters  kept  at  1.5°C.  for  at  least  one  hundred 
days. 

^Report  of  State  Board  of  Health  of  ^lass.,  1905,  p.  455. 


BACTERIOLOGY  OF  THE  OYSTER.  63 

The  important  conclusion  to  be  drawn  from  this  series  of  experi- 
ments is  that  under  the  conditions  of  the  experiment  bacteria  of  the 
B.  coh  group  do  not  materially  increase  or  decrease  in  oysters  in  the 
shell  during  storage. 

CLEANSING  OF  POLLUTED  OYSTERS. 

As  soon  as  the  etiological  connection  between  oysters  and  certain 
epidemics  of  typhoid  and  gastro-enteritis  was  firmly  established,  the 
question  at  once  arose  as  to  how  long  a  time  it  would  take  oysters 
known  to  be  polluted  to  free  themselves  from  sewage  organisms  after 
they  had  been  removed  to  water  free  from  sewage  contamination. 

Klein^  put  oysters  into  tanks  in  the  laboratory  and  infected  them 
withB.  typhosus.  About  one-third  of  the  water  was  removed  every 
day  and  replaced  with  clean  sea  water.  Oysters  were  removed  at 
various  intervals  and  examined  for  B.  typhosus.  The  experiments 
were  repeated  several  times  and  B.  typhosus  was  isolated  at  the  end 
of  the  experiment  in  every  case.  The  various  experiments  were  con- 
cluded on  the  seventh,  ninth,  fourteenth,  sixteenth,  seventeenth  and 
eighteenth  day  after  infection.  The  bacilli  were  isolated  from  the 
sea  water  twenty-one  days  after  the  beginning  of  the  experiment. 
Of  course,  these  experiments  did  not  approximate  natural  con- 
ditions and  so  we  can  draw  no  definite  conclusions  from  them  regard- 
ing the  length  of  time  necessary  for  oysters  to  rid  themselves  of  these 
bacteria  when  taken  from  polluted  areas  and  re-layed  in  water  free 
from  pollution. 

Herdmann  and  Boyce^  tried  the  experiment  of  infecting  oysters 
artifically  with  large  numbers  of  B.  typhosus  and  then  subject- 
ing them  "to  a  running  stream  of  pure  clean  sea  water." 
Eighteen  oysters  were  infected  and  examined  at  different  intervals 
varying  from  one  to  seven  days.  Only  the  stomach  contents  were 
examined  and  considerable  allowance  must  be  made  for  this,  for  the 
writer  has  sho^vn  in  another  part  of  this  paper  that  the  number  of 
bacteria  contained  in  the  stomach  are  quite  insignificant  compared 
with  the  number  in  the  shell  liquor  and  on  the  body  of  the  oyster. 

In  three  of  the  eighteen  oysters  examined  which  had  washed  for 
three,  five  and  seven  days,  respectfully,  no  typhoid  bacilli  were 
found.     In  the   other  fifteen   oysters  examined   B.   typhosus   was 

'Relation  of  Oysters  and  Disease,  Supplement  to  the  Report  of  the  Medical  Officer  to  the  Local 
Government  Board,  1893. 
^Loc.  cit. 


64  BACTERIOLOGY  OF  THE  OYSTER. 

found  in  varying  numbers.  Herdmann  and  Boyce  sum  up  the 
matter  as  follows:  "The  result  was  definite  and  uniform;  there  was 
a  great  diminution  or  total  disappearance  of  B.  typhosus  in  from  one 
to  seven  days." 

Johnstone^  took  oysters  known  to  be  polluted  and  transferred 
to  the  purest  water  available.  He  found  under  the  conditions  of 
the  experiment  that  four  days  was  a  sufficient  period  of  quarantine, 
since  after  that  time  no  further  cleansing  took  place,  because  the 
water  of  the  locality  was  not  entirely  free  from  sewage  contamination. 

Phelps  in  this  country^  found  that  only  two  to  four  days  was 
necessary  for  polluted  oysters  to  cleanse  themselves  when  transferred 
to  clean  water. 

In  1913,  Fabre-Domergue^  read  a  paper  before  the  Academic  de 
Medicine  in  which  he  recommended  the  placing  of  polluted  oysters  in 
basins  fed  by  filtered  water  and  removed  often  enough  to  insure  com- 
plete evacuation  of  the  Hquid  contained  in  the  shells  and  in  the 
digestive  tract.  From  his  results  he  considers  it  an  established  fact 
that  this  procedure  eliminates  all  pathogenic  bacteria  from  the  mol- 
luscs in  six  or  seven  days. 

Field*  says:  "These  (oysters)  get  bacteria  from  the  waters  filled 
with  waste  and  sewage,  and  it  takes  them  at  least  seventy-two 
hours  to  free  themselves  from  these  impurities  that  they  have  taken 
in  from  the  waters  of  the  different  harbors."  Field  does  not  say  upon 
what  evidence,  if  any,  this  statement  is  based.  But  he  adds  that 
in  Massachusetts  a  law  has  been  passed  requiring  such  polluted 
oysters  to  be  transferred  to  clean  water  and  allowed  to  remain  for 
four  weeks  before  offered  for  sale. 

The  writer's  own  experiments  on  the  cleansing  of  polluted  oysters 
confirm  in  part  the  work  cited  above.  It  appears  that  the  rapidity 
with  which  sewage  bacteria  are  eliminated  is  influenced  to  quite  a 
large  extent  by  the  temperature.  If  the  water  is  warm,  say  around 
20° — 25  C.  the  oysters  remain  open  probably  most  of  the  time. 
As  this  is  about  the  optimum  temperature  for  the  most  rapid  growth 
and  development  of  the  oyster,  it  is  also  the  temperature  at  which 
the  oyster  is  most  active.  The  ciliary  motion  is  more  rapid  than  at 
lower  temperatures   which   would   increase   the   amount   of   water 

iJour.  of  Hyg    IX,  1909. 

*Jo'.ir.  Am.  Public  Health  Assn.,  Vol.  1,  1911,  30-5. 

^Cited  in  Jour.  Am.  Med.  Asso.,  LXI,  1913,  134. 

Report  of  Proceedings  of  3rd  Am.  Convention  of  Nat.  Asso.  of  Shellfish  Commission,  p.  34. 


BACTERIOLOGY  OF  THE  OYSTER.  65 

filtered  through  the  gills  and  so  increase  the  amount  of  "wash  water" 
for  carrying  away  the  bacteria.  Also  the  ciliary  motion  in  the 
alimentary  canal  would  be  hastened  and  so  the  organisms  contained 
therein  would  be  more  quickly  disposed  of.  Further,  the  capacity  of 
the  oyster  to  digest  and  assimulate  bacteria  would  be  at  its  height  at  a 
temperature  at  which  the  cells  are  most  active.  Hence,  the  optimum 
temperature  for  the  growth  and  development  of  the  oyster  we  would 
expect  to  be  the  period  at  which  all  contaminating  organisms  would 
be  eliminated  most  rapidly.  As  the  temperature  lowers  the  activities 
of  the  oyster  lessen  accordingly.  Further,  while  above  20°C.  the 
oyster  has  its  valves  open  most  of  the  time,  as  the  temperature  is 
lowered  the  oyster  is  more  and  more  inclined  to  keep  its  valves  closed 
for  longer  and  longer  periods.  This  would  prevent  the  mechanical 
effect  of  the  filtered  water  in  carrying  away  the  bacteria.  This 
mechanical  effect  is  very  important  for  the  writer  has  shown  in 
another  part  of  this  paper  that  bacteria  pass  through  the  gills  with 
the  filtered  water  very  rapidly.  Further,  the  activity  of  the  cells 
concerned  in  the  digestion  of  bacteria  would  also  be  less  active  and 
also  the  antagonism  between  different  species  of  bacteria  would  be 
lessened.  So  it  is  seen  that  at  lower  temperature  the  tendency  would 
be  for  oysters  to  eliminate  bacteria  more  slowly  than  at  higher 
temperatures.  Various  opinions  have  been  expressed  regarding  the 
temperature  at  which  oysters  ''hibernate"  or  close  their  shells  and 
remain  closed  due  to  the  low  temperature  of  the  water.  The  theory 
of  "hibernation"  of  the  oyster  was  first  proposed  by  Gorham^  to 
explain  the  results  obtained  in  his  investigation  of  the  sanitary  con- 
ditions of  the  oyster  beds  of  Narragansett  Bay.  The  temperature 
at  which  this  phenomenon  is  supposed  to  take  place  is  a  little  above 
0°C.  So  far  as  the  writer  is  aware  no  experimental  work  of  an  exact 
nature  has  been  done  to  substantiate  or  disprove  this  theory,  but 
from  personal  observation  the  writer  is  led  to  suspect  that  the 
temperature  at  which  the  oyster  closes  its  shell  for  a  relatively 
long  period  is  considerably  higher  as  will  appear  from  one  of  the 
experiments  detailed  below. 

On  the  other  hand,  experiments  to  be  detailed  later  under  the 
hibernation  of  oysters  seem  to  show  that  oysters  do  open  and  are 
active  at  temperatures  only  one  to  two  degrees  above  0°C.  It 
appears  that  when  the  temperature  is  low  oysters  will  close  their  shell 

1(1)  Rep.  of  Commissioners  of  Shell  Fisheries  of  R.  I.,  1910.  (2)  Seasonal  Variation  in  the 
Bacterial  Content  of  Oyster.s,  Jour.  Am.  Pub.  Health,  Jan.,  1912. 


66  BACTERIOLOGY  OF  THE  OYSTER. 

for  sometime,  but  not  for  indefinite  periods.  It  also  appears  that 
the  closure  of  the  shell  is  not  due  to  cold  rigor  or  loss  of  control  of  the 
adductor  muscle,  for  at  a  temperature  of  1.5°C.  the  oyster  can  open 
and  close  its  shell  with  the  same  ease  as  at  higher  temperatures. 

In  the  following  experiments  only  the  shell  liquor  was  used.  The 
method  of  examination  of  the  oysters  was  the  procedure  recom- 
mended by  the  Second  Progress  Report  of  the  Committee  on  Standard 
Methods  of  Shellfish  Examination  of  the  American  Public  Health 
Association.  The  medium  used  was  lactose-peptone  bile  and  the 
tubes  were  inoculated  in  duplicate.  The  tubes  were  examined  every 
twenty-four  hours  for  three  days.  If  ten  per  cent,  or  more  of  gas 
appeared  during  this  time,  it  was  considered  to  show  the  presence  of 
intestinal  bacteria.  Unfortunately  in  the  first  experiment  the 
investigation  had  to  be  discontinued  after  November  29th,  so  that 
we  have  only  the  results  extending  over  12  days. 

Experiment  I. 

November  16,  1912,  about  a  bushel  of  polluted  oysters  were  taken 
from  the  Providence  River  and  the  following  day  were  transferred  to 
Wickford  Harbor.  They  were  laid  upon  clean  sandy  bottom  on  the 
edge  of  the  channel  and  were  well  separated  to  allow  free  access  of 
water.  The  temperature  of  the  water  at  the  time  of  taking  the 
oysters  was  14°C.  The  average  of  the  maximum  and  minimum 
temperature  at  Wickford  for  November  16  and  17  was  6.5°C. 
A  sample  of  the  oysters  was  taken  at  the  time  they  were  placed  in 
Wickford  Harbor  and  the  analysis  showed  a  score  on  fifteen  oysters  of 
870.  Samples  were  shipped  to  the  laboratory  every  day  until 
November  29th.  These  were  analyzed  immediately  so  that  only 
three  or  four  hours  elapsed  between  the  time  of  collecting  the  sample 
and  the  time  of  analysis.  The  following  table  shows  the  results  of 
the  analysis  of  fifteen  oysters  on  the  different  days.  The  temperature 
is  the  average  of  the  maximum  and  minimum  temperature  as  recorded 
at  the  lobster  hatchery  of  the  Inland  Fish  Commission  which  was 
located  nearby. 


BACTERIOLOGY    OF    THE    OYSTER. 


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BACTERIOLOGY    OF   THE    OYSTER. 


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BACTERIOLOGY   OF   THE    OYSTER. 


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BACTERIOLOGY   OF   THE    OYSTER. 


71 


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72  BACTERIOLOGY  OF  THE  OYSTER, 

Unfortunately  the  experiments  could  not  be  continued  and  so  we 
cannot  say  whether  the  apparent  cleansing,  which  appeared  on  the 
last  two  days,  especially  on  the  last  day,  was  due  to  a  fortunate 
selection  of  oysters  or  was  the  indication  of  a  real  elimination  of  the 
intestinal  bacteria.  The  writer  is  led  to  believe  that  the  oysters 
had  just  begun  to  open  and  so  allowed  the  bacteria  to  be  washed  out. 
The  low  temperature  of  the  water  slowed  the  metabolic  processes  of 
the  oyster  and  so,  as  food  and  oxygen  were  not  needed  in  so  great 
quantities,  an  oyster  could  maintain  itself  for  sometime  without 
renewing  its  supply.  As  soon  as  the  supply  was  exhausted,  however, 
the  oyster  opened  its  shell. 

This  investigation  shows  that  under  the  conditions  of  the  experi- 
ment with  a  temperature  between  7.2°C.  and  5°C.  a  period  of  twelve 
days  is  not  sufficient  to  allow  oysters  to  free  themselves  from  intestinal 
bacteria. 

Experiment  II. 

May  13,  1913,  about  a  bushel  of  polluted  oysters  were  taken 
from  Providence  River  and  transferred  to  the  same  location  in 
Wickford  Harbor  as  in  the  previous  experiment. 

The  water  of  Wickford  Harbor  at  the  place  where  the  oysters  were 
put  down  was  tested  by  the  lactose-peptone-bile  presumptive  test  and 
no  sewage  organisms  were  found.  The  methods  and  conditions  of 
the  experiment  were  the  same  as  in  the  previous  experiment  except 
that  ten  oysters  w^ere  used  instead  of  fifteen.  The  sanitary  condition 
of  the  oysters  at  the  time  of  transplantation  and  on  two  subsequent 
occasions  is  shown  in  the  following  table: 


BACTERIOLOGY    OF   THE    OYSTER. 


73 


Tables  Showing  Results  of  Analysis  of  Ten  Oysters  from  a  lot  of  Polluted  Oysters  which  had 
been  put  into  Relatively  Unpolluted  Water  at  Wickford,  May  13,  1913. 
Date,  May  13,  1913. 


Average  Temperature. 

Dilution  op 
Shell  Liquor. 

No.  of  Oyster. 

1 

2 

3        I        4 

5 

6 

7 

8 

9 

10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+       + 
+       + 

0      0 

+       + 
+       + 
+       + 

+       + 
+       + 

+      0 

+    + 

0      0 
0      0 

+   + 
+   + 

+      0 

+   + 
+  + 

+      0 

+   + 
+  + 
+  + 

+   + 
+   + 

0      0 

+   + 

+      0 
0      0 

+   + 

0      0 
0      0 

Score,  460. 


Date,  May  17,  1913. 


Average  Temperature, 

12.7»C. 

Dilution  of 
Shell  Liquor. 

.  No.  of  Oyster. 

1        1        2 

3 

4 

5 

6 

7 

8 

9 

10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+  +;+  + 

+    o!o     0 

0       0    0       0 

0      0 

+       + 
+       + 
0      0 

+       + 

0      0 
0       0 

+      0 
+      0 
0      0 

+      0 
0      0 
0      0 

+    + 

+      0 
0      0 

+  + 

+   + 

+      0 

+     4- 
0      0 
0      0 

Score,  73. 


Date,  May  22,  1913. 


Average  Temperature  14.7°C. 

Dilution  of 
Shell  Liquor. 

No.  of  Oyster. 

1 

^ 

3 

4 

5        I       6 

7 

8 

9 

1       10 

\ 

Ic.c 

1-lOc.c 

1-lOOc.C 

+ 
+ 
0 

oj+ 

0   + 
0   0 

+ 

0 
0 

+ 

0 
0 

+ 

0 
0 

+      + 
0      0 
0      0 

+    + 
+   + 

0      0 

+    + 

0      0 
0      0 

0       0 
0      0 
0      0 

+   + 

0      0 
0      0 

+ 

0 
0 

+ 

0 
0 

+   + 

0      0 
0      0 

Score,  28. 


74  BACTERIOLOGY  OF  THE  OYSTER. 

From  the  table  it  is  seen  that  at  the  beginning  of  the  experiment 
there  were  on  the  average  forty-six  B.  coh  per  cubic  centimeter  of 
oyster  juice.  After  a  period  of  four  days  this  number  had  dropped  to 
an  average  of  7.3  per  cubic  centimeter  and  after  a  period  of  nine  days 
the  number  had  still  further  decreased  so  that  there  were  on  the  average 
only  2.8  B.  coli  per  c.c.  of  the  oyster  juice.  This  shows  that  under  the 
conditions  of  the  experiment,  oysters  which  contained  46  B.  coli  per 
cubic  centimeter  can  in  nine  days  free  themselves  from  B.  coli  to 
such  an  extent  that  there  remains  only  2.8  B.  coli  pr  cubic  centimeter. 
This  is  well  within  the  standard  adopted  by  the  Bureau  of  Chemistry 
which  allows  oysters  to  be  shipped  in  interstate  commerce  which 
contain  4.6  B.  coli  per  cubic  centimeter  of  shell  liquor. 

Experiment  III. 

On  November  8,  1913  a  bushel  of  oysters  were  taken  from  Provi- 
dence River  and  transplanted  to  Wickford.  These  oysters  were  put 
into  two  galvanized  iron  baskets  and  hung  into  the  water  from  the 
floor  of  the  Beacon  Oyster  Co.  These  oysters  were  suspended  in  the 
water  near  the  edge  of  the  channel  and  located  only  a  few  yards  from 
the  place  where  the  oysters  in  the  two  previous  experiments  were 
placed.  A  sample  of  ten  oysters  was  taken  from  this  lot  and  carried 
to  the  laboratory  for  analysis.  These  ten  oysters  were  found  to  be 
badly  polluted  and  had  a  score  of  640.  Samples  were  sent  to  the 
laboratory  and  analyzed  on  November  10,  12,  14,  17,  19,  21  and  24. 
The  methods  of  analysis  were  the  same  as  in  the  previous  experi- 
ments with  two  exceptions.  The  1-10  c.c.  and  1-100  c.c.  dilutions 
were  made  in  duplicate,  while  the  one  cubic  centimeter  samples  were 
only  inoculated  singly.  The  oyster  liquor  was  drained  into  glass- 
stoppered  bottles  which  were  graduated  so  that  the  amount  of  liquor 
could  be  read  off  in  cubic  centimeters.  An  equal  amount  of  sterile 
one  per  cent,  sodium  chloride  solution  was  added  and  the  bottle 
shaken  vigorously  one  hundred  times.  One  cubic  centimeter  of  this 
mixture  was  used  for  the  first  inoculation  and  to  make  the  proper 
dilutions.  As  a  result  the  quantities  as  given  in  the  table  are  for  the 
mixture  of  shell  liquor  and  salt  solution.  The  amount  of  shell  liquor 
in  the  dilutions  is  not  1  c.c,  1-10  c.c.  and  1-100  c.c,  but  }/2  c.c, 
1-20  c.c.  and  1-200  c.c.  But  as  ten  oysters  were  used  the  result 
equals  an  analysis  of  five  oysters  where  1  c.c,  1-10  c.c.  and  1-100  c.c 
samples  of  the  shell  liquor  were  used.     For  comparative  results, 


BACTERIOLOGY    OF    THE    OYSTER. 


75 


however,  it  does  not  matter  what  quantity  we  use  provided  we  use 
the  same  amount  every  time.  The  following  table  shows  the  results 
of  the  examination  on  the  different  days. 

Table  Showing  the  Results  of  Anahjsis  of  Polluted  Oysters  which  were  put  into  Compar- 
atively Uncontaminaied  Water  at  Wickford,  November  8,  1913. 
Date,  November  8,  1913. 


Quantity  of 
Shell   Liquor 
AND  Salt  Solu- 

Average  Tempebature. 

No.  of  Oyster. 

1 

2 

3 

4 

5 

« 

7 

8 

9 

10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+       +'+       + 

0      0+     + 

+ 
+       + 
+       + 

+ 
+      + 
+      0 

+       1       + 
+       +'+       + 

+      0  +     + 

+ 
+       + 
+      0 

+ 
+      + 
0      0 

+ 
+   + 

+      0 

+ 
+   + 

0       0 

Score,  730. 


Date,  November  10,  1913. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 

Average  Temperature 

11.1°C. 

No.  of  Oyster. 

1        !        2 

3 

4 

5        1        6 

7 

8 

9 

10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+    1     0 

+      0j+      0 
0       OiO       0 

1 

+ 

+      0 
0       0 

+ 

0      0 
0      0 

+  +1+  + 

+      000 

+ 
+   + 

0       0 

+ 
+   + 

0      0 

+ 

+       0 
0      0 

+ 

+      0 
+      0 

Score,  190. 


Date,  November  12,  1913. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 

Average  Temperature  8.8°C. 

No.  of  Oyster. 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Ic.c 

0 

0 

0 

0 

+ 

+ 

+ 

+ 

+ 

+ 

1-lOc.c 

0        0 

0       0 

0      0 

+      0 

0      0 

0       0 

+      0 

0      0 

+      0 

+      0 

1-lOOc.c 

0        0 

+      0 

0      0 

0      0 

0      0 

+      0 

0      0 

0      0 

+      0 

0      0 

Score,  37. 


76 


BACTERIOLOGY    OF   THE    OYSTER. 


Date,  November  4,  1913. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 

Average  Temperature 

9.7°C. 

No.  of  Oyster. 

1 

2 

3 

4 

5 

6 

7       i       8 

9              10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+ 

0      0 
0      0 

+ 

+      0 
0      0 

+ 

0       0 
0      0 

0 
0      0 
0       0 

0 
+      0 
0       0 

+ 

0       0 
0        0 

+ 

0      0 
0       0 

+ 

0      0 
0       0 

+ 

0       0 
0      0 

+ 

0      0 
0       0 

Score,  10. 


Date,  November  17,  1913. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 

Average  Temperature  7.7°C. 

No.  of  Oyster. 

1               2 

3 

4 

5 

5 

7 

8 

9 

10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+ 

0      0 
0      0 

+ 

0      0 
0      0 

+ 

0      0 
0       0 

+ 

+      0 
0       0 

+ 

0       0 
0      0 

+ 

+      0 
0      0 

+ 

0      0 
0      0 

+ 

0      0 
0       0 

+ 

0       0 
0      0 

+ 

0        0 
0       0 

Score,  10. 


Date,  November  19,  1913. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 

Average  Temperature  7.3°C. 

No.  of  Oyster. 

1 

2 

3 

4 

5               6 

7 

8 

9 

10 

Ic.c 

1-lOc.c 

1-lOOc.c 

+ 
+      0 
0      0 

+ 

0      0 
0      0 

+ 

+      0 
0      0 

+ 
0      0 
0      0 

0 
0      0 
0      0 

+ 
+    + 

0      0 

+ 

0       0 
0      0 

0 
0      0 
0      0 

+ 

0       0 
0      0 

0 
+      0 
0      0 

Score,  19. 


BACTERIOLOGY    OF   THE    OYSTER. 
Date,  November  21,  1913. 


77 


Average  Temperature  10.5°C. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 
tion. 


No.  of  Oyster. 


' 

2 

3 

+ 

+    + 

+ 

.L 

+      0 

0       0 

0 

0 

0       0 

0       0 

0 

0 

4 

5 

6 

7 

8 

9 

0           + 

+ 

+ 

+ 

+ 

0      0    0       0 

+      0 

0      0 

0      0 

0       0 

0      0 

0       0 

0      0 

0       0 

0       0 

0       0 

10 


Ic.c 

1-lOc.c 

1-lOOc.c 


+ 
0      0 
0       0 


Score,  19. 


Date,  November  24,  1913. 


Quantity  of 
Shell  Liquor 
AND  Salt  Solu- 
tion. 


Average  Temperature  10.5°C. 


No.  of  Oyster. 


Ic.c 

1-lOc.c 

1-lOOc.c 


1                2 

3 

4 

5 

+    1     0 

0      0    0       0 
0      0    0       0 

+ 
+    + 

0       0 

+ 

0      0 
0       0 

+ 

+      0 
0      0 

6               7 

8 

9 

+           0 
0       0    0      0 
0       0    0      0 

1 

+ 

+   + 

+      0 

+ 

0       0 
0      0 

10 


+ 

0       0 
0       0 


Score,  28. 


-|-  =  positive  presumptive  test  for  B.  coli. 
0=:  negative  presumptive  test  for  B.  coli. 


It  is  seen  from  the  tallies  that  the  oysters  cleaned  themselves  as 
much  in  six  days  as  at  any  time.  The  samples  taken  on  the  day  of 
transporting  showed  on  the  average  twenty-three  B.  coli  per  cubic 
centimeter  of  oyster  juice.  Six  days  later  the  sample  showed  only  one 
B.  coli  per  cubic  centimeter  and  they  showed  no  further  cleansing 
after  ten  more  days.  The  fact  that  the  water  at  this  place  is  not 
entirely  free  from  sewage  contamination  probably  explains  why  the 
oysters  did  not  show  any  further  elimination  of  B.  coli,  apparently 
there  were  enough  B.  coli  in  the  water  to  maintain  a  small  number  in 
the  oyster  at  all  times. 


Condasions. 

In  one  experiment  with  a  temperature  averaging  9.7''C.  over  the 
period  of  the  investigation  the  oysters  showed  an  elimination  of  B. 


78  BACTERIOLOGY  OF  THE  OYSTER. 

coli  from  73  per  cubic  centimeter  to  one  per  cubic  centimeter  in  six 
days.  In  another  experiment  with  an  average  temperature  of  13°C. 
during  the  period  of  investigation  the  oysters  showed  an  eUmination 
of  B.  coh  from  an  average  of  46  B.  coh  per  cubic  centimeter  to  7.3  B. 
coU  per  cubic  centimeter  in  four  days  and  to  2.8  per  cubic  centimeter 
of  shell  liquor  in  nine  days.  As  no  examination  was  made  between  the 
fourth  and  ninth  day,  it  is  quite  possible  that  the  limit  of  possible 
elimination  was  reached  sometime  before  the  ninth  day.  No  doubt 
an  examination  on  the  sixth  or  seventh  day  would  have  shown  a  B. 
coli  content  sufficiently  low  to  pass  the  standard  set  by  the  Bureau  of 
Chemistry  of  the  Federal  Government. 

In  another  experiment  in  November,  1912,  with  an  average  tem- 
perature 5.4°C.  twelve  days  was  not  sufficient  to  eliminate  B.  coli  to 
any  appreciable  extent.  The  examination  on  the  twelfth  day  showed 
a  very  marked  decrease  in  the  number  of  B.  coli,  but  as  no  subsequent 
examinations  were  made  it  is  not  possible  to  say  with  authority 
whether  this  was  the  beginning  of  an  elimination  process  or  not, 
though  the  writer  is  led  to  believe  such  was  the  case.  The  interesting 
feature  of  this  experiment  is  that  no  elimination  took  place  in  nine 
days,  while  in  the  other  two  experiments  a  very  marked  reduction 
took  place  in  six  days  in  one  case  and  in  five  and  nine  days  in  the  other 
case. 

These  sets  of  experiments  seem  to  throw  some  light  upon  the  so- 
called  hibernation  of  the  oyster.  With  an  average  temperature  of 
13°C.  in  one  case  and  9.7°C.  in  the  other  the  oysters  opened  and  began 
to  eliminate  B.  coli  almost  immediately,  but  in  the  first  experiment 
with  an  average  temperature  of  5.4°C.  no  reduction  in  B.  coli  was 
found  until  the  twelfth  day.  These  experiments  lead  the  writer  to 
beheve  that  when  the  temperature  of  the  water  is  somewhere  between 
9°C.  and  5°C.  oysters  close  their  shells  for  a  longer  or  shorter  period. 
But  from  experiments  detailed  elsewhere,  the  writer  believes  that 
there  is  no  time  above  0°C.  when  oysters  close  their  shells  for  an 
indefinite  period.  The  length  of  time  that  oysters  remain  closed  is  in 
inverse  proportion  to  the  temperature  which  determines  the  rapidity 
of  the  metabolic  processes  going  on  within  the  oyster. 

EXPERIMENTS  ON  THE  HIBERNATION  OF  THE  OYSTER. 

The  so-called  hibernation  of  oysters  has  attracted  much  attention 
during  the  last  four  years.     The  theory  that  oysters  close  their  shells 


BACTERIOLOGY  OF  THE  OYSTER.  79 

when  the  temperature  of  the  water  approaches  0°('.  was  first  put 
forward  by  Gorham  in  1910;^  to  explain  certain  bacteriological  find- 
ings in  Providence  River  oysters.  It  was  found,  that  during  the 
warmer  months  the  oysters  in  certain  parts  of  the  river  were  badly 
polluted,  but  in  January,  with  the  temperature  of  the  water  around 
0°C.,  the  oysters  were  found  free  from  colon  bacilli.  In  order  to 
explain  this  phenomenon  Gorham  advanced  the  theory  that  when  the 
temperature  of  the  water  approaches  0°C.  the  oyster  closes  its  shell 
and  remains  closed  until  the  temperature  of  the  water  begins  to  rise 
and  then  it  opens  its  shell  and  resumes  its  normal  activity.  This 
period  was  called  its  "Hibernation  Period."  A  little  later  Pease, ^ 
Field,  of  the  Massachusetts  Fish  and  Game  Commission,  and  others 
advanced  a  similar  idea.  So  far  as  the  writer  is  aware,  however,  no 
experiments  have  been  tried  to  confirm  or  deny  this  theory.  The 
experiments  of  the  writer  cited  elsewhere  on  the  cleansing  of  polluted 
oysters  seem  to  show  that  oysters  do  remain  closed  for  several  days 
with  a  temperature  of  about  5°C.  But  in  order  to  throw  further 
light  upon  the  matter  the  following  experiments  were  tried. 

Experiment  I. 

January  12,  fourteen  oysters  were  placed  in  sea  water  which  had 
been  inoculated  with  a  pure  culture  of  B.  coli.  The  oysters  were 
left  in  the  sea  water  a  day  and  a  night.  They  were  removed  January 
13th,  and  the  outside  of  the  shells  scrubbed  thoroughly  with  a  stiff 
brush  and  running  tap  water  and  were  then  put  into  a  strong  solution 
of  calcium  hypochlorite  for  one-half  hour  and  stirred  up  about  once 
a  minute.  They  were  then  put  into  7%  formalin  for  the  same  length 
of  time  and  stirred  with  a  glass  rod  for  a  few  seconds  at  about  one 
minute  intervals.  They  were  then  washed  for  a  considerable  time  in 
fast  running  tap  water,  temperatures  between  7°C.  and  8°C.,  and 
stirred  at  intervals  of  two  or  three  minutes.  The  oysters  were  then 
taken  (Jan.  13),  to  a  cold  storage  room  of  the  Merchant's  Cold 
Storage  and  Warehouse  Co.,  Providence,  and  put  into  storage  at 
34°F.  (about  1.1°C.)  The  temperature  of  the  room  is  maintained 
constant  throughout  the  year  and  is  never  allowed  to  vary  more  than 
.5°F.     The  next  day  sterile  sea  water  which  had  been  kept  in  the 

^(l)  Report  of  Commissioners  of  Shell  Fisheries  of  R.  I.,  1910.  (2)  Seasonal  Variation  in  the 
Bacterial  Content  of  Oysters,  Am.  Jour.  Pub.  Health,  II,  1910,  24. 

^Some  Bacteriological  Problems  in  the  Oyster  Industry,  The  Fishing  Gazette,  28,  1911,  865. 
July  15. 


80  BACTERIOLOGY    OF    THE    OYSTER. 

room  for  several  days  was  poured  into  the  dishes  until  it  covered  the 
oysters.  Immediately  after  the  oysters  were  covered  five  samples 
of  two  cubic  centimeters  each  were  taken  from  each  dish  and  inocu- 
lated into  bile  tubes  and  incubated  at  37°C.  for  18  hours.  Every  tube 
showed  gas.  January  22,  the  oysters  were  examined  in  the  dishes 
and  it  was  found  that  four  were  closed  tightly,  five  were  open  widely 
enough  to  be  seen  as  they  lay  in  the  dishes  and  the  other  seven  were 
found  to  be  slightly  open.  The  opening  of  these  last  seven  was  not 
perceptible  to  the  eye,  but  upon  taking  them  out  and  squeezing  them 
one  could  hear  a  "squashy"  sound,  showing  that  they  were  not  firmly 
closed.  Apparently  the  five  oysters  that  were  open  had  lost  their 
sensitiveness,  for  they  would  not  remain  closed  when  the  valves  were 
pressed  together.  The  mechanical  stimulation  of  the  gills  and  mantle 
was  not  tried.  The  oysters  were  observed  on  several  days  until 
February  2nd  and  it  was  found  that  some  of  the  oysters  that  had  been 
firmly  closed  at  first  had  opened  and  vice  versa. 

The  oysters  were  not  observed  again  until  March  23.  It  was  found 
that  two  of  the  oysters  in  one  dish  were  open  and  dead.  Two  others 
were  wide  open  but  closed  immediately  when  touched.  These  two 
oysters  were  brought  to  the  laboratory  and  put  into  a  dish  of  sterile 
sea  water  and  observed  for  several  days.  They  were  just  as  active 
as  oysters  freshly  brought  from  the  beds.  They  were  then  tested  for 
B.  coli.  Both  oysters  showed  gas  in  1-100  c.c.  of  shell  liquor.  These 
tubes  were  plated  in  litmus-lactose-agar  and  typical  colon  colonies 
were  found  in  the  plates  from  one  oyster,  but  not  from  the  other. 
This  showed  that  B.  coli  can  live  under  such  condition  for  at  least 
sixty-nine  days. 

The  remaining  oysters  were  again  examined  April  24,  one  hundred 
days  after  they  were  put  into  storage.  Five  of  the  oysters  were 
apparently  living,  while  the  others  were  dead.  These  five  were 
brought  to  the  laboratory  and  examined.  It  was  found  that  three  were 
closed  tightly,  while  the  other  two  appeared  a  little  "weak."  One 
of  the  tightly  closed  oysters  was  put  into  a  dish  of  sea  water  and  it 
soon  opened  like  an  oyster  removed  only  recently  from  its  natural 
element.  When  the  shell  was  touched  it  would  close  immediately, 
though  its  movements  were  not  so  vigorous  as  those  of  an  oyster 
taken  directly  from  the  water.  When  the  gills  and  mantle  were 
touched  with  a  wire  it  did  not  respond  readily.  Apparently  its 
tactile  sensations  were  not  very  acute,  although  after  repeated 
stimulations  it  closed  and  gripped  the  wire  so  that  it  took  considerable 


BACTERIOLOGY   OF   THE    OYSTER.  81 

strength  to  pull  it  out.  The  writer  has  noticed  that  oysters  which 
have  been  removed  from  sea  water  for  some  time  require  a  great  deal 
of  stimulation  to  make  them  close  again,  though  after  they  have  been 
open  for  a  time  they  react  immediately.  It  may  be  that  the  tango- 
receptors  are  very  much  dulled  or  that  the  desire  for  oxygen  is  stronger 
than  the  sense  of  self-protection. 

The  other  four  oysters  were  opened  with  the  proper  precautions 
and  the  mixed  shell  liquor  and  the  "washings"  from  the  body  were 
inoculated  into  bile  tubes.  Two  of  the  oysters  were  normal  in 
appearance  and  exceptionally  plump.  The  other  two  showed  slight 
evidences  of  decomposition.  All  the  tubes  from  three  of  the  oysters 
showed  gas  and  typical  B.  coli  colonies  were  isolated  on  litmus-lactose- 
agar  plates.  Further  identification  was  not  regarded  as  necessary. 
The  tubes  inoculated  from  the  third  oyster  showed  no  gas  after  three 
days  incubation. 

Experiment  IL 

Seven  oysters  were  obtained  fresh  from  the  water  and  impregnated 
with  a  solution  of  azolitmin  in  sea  water.  They  were  then  washed 
thoroughly  with  a  stiff  brush  in  running  water  and  immersed  in 
chromic  acid  for  a  few  seconds  and  then  washed  again.  All  the 
color  was  removed  in  this  manner.  January  29  they  were  placed 
in  tumblers  and  put  into  cold  storage  at  34°F.  They  were  left  over 
night  to  acquire  the  same  temperature  as  the  room  and  then  the 
tumblers  were  filled  with  sea  water.  The  dishes  were  watched  to  see 
if  any  color  had  escaped  from  the  oysters.  February  2  a  slight 
coloration  was  found  in  the  bottom  of  two  of  the  tumblers,  but  this 
did  not  appear  to  increase  for  several  days.  The  oysters  were  not 
examined  again  until  March  23rd.  The  color  had  disappeared  from 
the  two  tumblers  that  had  previously  been  discolored.  It  was 
observed,  however,  that  the  water  in  the  tumblers  was  not  entirely 
clear.  There  was  a  sediment  in  the  bottom  of  the  tumblers  that 
resembled  the  bits  of  mucus  thrown  off  by  oysters.  One  of  the 
oysters  was  taken  to  the  laboratory  and  placed  in  sea  water.  It 
soon  opened,  but  did  not  contain  any  color.  It  was  as  active  as  a 
normal  oyster.  Some  of  the  mucus  thrown  out  by  the  oyster  had  a 
purplish  color  which  had  been  stained  with  azolitmin. 

April  17  the  remaining  six  oysters  were  examined.  It  was  found 
that  three  of  the  oysters  were  open  and  the  other  three  closed.     Covers 


82  BACTERIOLOGY  OF  THE  OYSTER. 

were  fitted  to  all  the  dishes  and  during  the  process  two  of  the  oysters 
closed.  The  other  one  remained  open  even  after  reaching  the 
laboratory.  After  the  cover  was  removed  and  the  shell  touched  with 
a  glass  rod  it  closed  immediately.  A  heavy  precipitate  was  found  on 
the  bottom  of  each  of  the  dishes  and  a  great  deal  of  mucus  was  seen 
in  suspension.  This  matter  could  not  have  come  from  the  outside 
of  the  oyster,  because  they  were  thoroughly  cleaned  before  the 
experiment  began.  There  is  no  question  but  what  the  oyster  had 
opened;  three  were  found  open  and  two  of  them  closed  immediately 
upon  being  agitated.  The  other  three  must  have  opened  in  order 
to  discharge  so  much  mucus,  but  had  closed  again  of  their  own 
accord  at  34°F. 

Two  oysters  were  infected  with  B.  coli  and  put  into  dishes  in  cold 
storage  January  20.  The  dishes  were  later  found  to  be  cracked  and 
the  water  leaked  out.  These  two  oysters  were  brought  to  the 
laboratory  April  17.  One  was  put  into  a  dish  of  sea  water,  while 
the  other  was  opened  and  two  cubic  centimeters  of  the  juice  was 
inoculated  into  each  of  four  bile  tubes.  Gas  appeared  in  each  tube 
and  typical  B.  coli  was  isolated  on  litmus-lactose-agar  plates.  This 
was  eighty-seven  days  after  infection.  The  other  oyster  opened 
before  morning,  but  was  apparently  dead  for  it  would  not  respond  to  a 
mechanical  stimulation  of  its  gills  and  mantle.  When  opened  both 
oysters  appeared  plump  and  in  prime  condition.  From  their  appear- 
ance they  could  not  have  been  told  from  oysters  freshly  caught. 

From  these  experiments  the  writer  believes  that  oysters  do  close 
their  shells  for  varying  periods,  depending  upon  the  temperature. 
Whether  they  close  their  shells  under  natural  conditions  when  the 
temperature  falls  around  0°C.  no  one  has  determined.  That  they  do 
not  lose  control  of  their  adductor  muscles  is  demonstrated  in  both 
experiments.  The  writer  is  lead  to  believe  that  there  is  no  definite 
period  at  which  this  phenomenon  can  be  said  to  begin.  Mitchell 
in  an  unpublished  observation  states  that  with  a  temperature  below 
20°C.  oysters  get  "nervous"  and  will  close  upon  the  slightest  provo- 
cation and  remain  closed  for  fairly  long  periods.  It  appears  that  at 
this  temperature  the  irritability  of  the  oyster  is  much  increased. 

These  experiments  lead  one  to  conclude  that  the  so-called  period 
of  hibernation  of  the  oyster  is  a  relative  term.  The  length  of  time 
that  they  remain  closed  depends  upon  the  temperature  which  deter- 
mines the  rapidity  of  the  oxidative  and  other  metabolic  processes  of 
the  oyster.     An  oyster  will  remain  closed  as  long  as  its  supply  of 


BACTERIOLOGY  OF  THE  OYSTER.  83 

food  and  oxygen  remains  sufficient  and  the  lower  the  temperature 
the  longer  this  period  will  be.  The  oyster  does  not  close  on  account 
of  "rigor  frigoris,"  for  the  control  of  the  adductor  muscle  is  still  very 
marked  at  a  temperature  of  l.l'^C.,  and  is  scarcely  distinguishable 
from  normal. 

Mitchell/  in  an  extended  study  of  the  oxygen  requirements  of 
shellfish  states  as  one  of  his  conclusions  that  "oysters  of  medium 
sizes,  at  temperatures  between  19°  and  28°C.,  used  from  7  to  35  deci- 
milligrams  of  oxygen  per  hour  per  100  grams  of  entire  weight.  The 
amount  varies  with  the  temperature,  so  far  as  experiments  show, 
according  to  simple  relationship,  so  that  the  curve  approximates 
a  straight  line."  .  .  .  "The  common  clam  (Mya  Arenaria) 
shows  a  higher  oxygen  requirement  than  the  oyster." 

The  theory  of  hibernation  which  the  writer  has  advanced  appears 
to  be  in  harmony  with  the  experiments  of  Mitchell  on  the  oxygen 
requirements  of  oysters.  The  lower  the  temperature  the  less  the 
amount  of  oxygen  used.  But  no  matter  what  the  temperature  so 
long  as  the  oyster  is  living  it  needs  a  certain  amount  of  oxygen  to 
carry  on  its  oxidative  processes.  When  the  amount  available  within 
its  shell  is  exhausted,  it  will  open  to  renew  its  supply. 

The  statements  of  practical  oyster  growers  also  leads  to  the  same 
conclusion.  It  is  said  that  oysters  from  Narragansett  Bay  in  February 
cannot  be  shipped  very  far  in  the  shell,  because,  as  the  oyster  men  say, 
they  will  "cluck,"  that  is,  open  their  shells  and  allow  the  shell  liquor 
to  run  out.  The  explanation  no  doubt  is  that  during  the  "zero 
weather"  of  January,  the  oysters  are  closed  and  as  their  oxygen 
requirements  under  the  circumstances  are  small  they  can  remain 
closed  for  sometime  without  exhausting  the  supply  available  in  the 
shell  liquor.  The  period  of  cold  weather,  however,  is  sufficiently  long 
perhaps  to  allow  the  oysters,  even  with  their  small  requirements,  to 
nearly,  if  not  quite  exhaust  the  available  supply  of  oxygen  within 
their  closed  shells.  The  result  is  that  in  February  when  they  are 
removed  to  the  opening  house  or  express  car  which  has  relatively  a 
much  higher  temperature  than  the  water  from  which  they  were  taken, 
the  metabolic  processes  of  the  oyster  are  greatly  increased  and 
there  is  a  demand  for  more  oxygen.  The  supply  within  the  shell, 
which  has  already  been  greatly  reduced,  is  quickly  used  up,  and 
consequently  the  oyster  opens  to  renew  its  supply. 

iThe  Oxygen  Requirements  of  Shellfish,  Bull.  U.  S.  Bureau  of  Fisheries,  XXXII,  1912,  209. 


84  BACTERIOLOGY  OF  THE  OYSTER. 

It  is  said  that  the  soft-shelled  clam  does  not  hibernate  during  the 
winter.  The  second  quotation  from  Mitchell's  paper,  namely,  that 
the  oxygen  requirement  in  the  common  clam  is  higher  than  in  the 
oyster  may  account  for  this  phenomenon.  The  sooner  the  available 
quantity  of  oxygen  is  used  up,  the  more  quickly  will  the  mollusc  open 
to  renew  its  supply. 

SUGGESTED  CHANGES  IN   STANDARD  METHODS  OF 
SHELLFISH  EXAMINATION. 

The  Second  Progress  Report  of  the  Committee  on  Standard 
Methods  of  Shellfish  Examination  recommends  that  "twelve  oysters 
of  the  average  size  of  the  lot  under  examination,  with  deep  bowls, 
short  lips  and  shells  tightly  closed,  shall  be  picked  out  by  hand  and 
prepared  for  transportation  to  the  laboratory."     .     .     . 

"Bacterial  counts  shall  be  made  of  a  composite  sample  of  each  lot 
obtained  by  mixing  the  shell  liquor  of  five  oysters."     .     .     . 

Under  the  heading  of  "Methods  of  Rating  Oysters  for  B.  coli," 
the  following  statement  is  made:  "The  following  values  shall  be 
assigned  to  the  presence  of  bacteria  of  the  B.  coli  group  in  each  of  the 
five  oysters  examined."  Then  follows  a  statement  and  illustration 
of  the  method  of  scoring  as  adopted  by  the  American  Public  Health 
Association.  It  is  clear  at  once  that  if  we  mixed  the  shell  liquor  of 
the  five  oysters  and  examined  it  as  a  composite  sample,  it  would  be 
impossible  to  assign  values  "to  the  presence  of  bacteria  of  the  B.  coli 
group  in  each  of  the  five  oysters  examined,"  for  the  composite  sample 
must  be  treated  as  the  juice  of  a  single  oyster.  It  is  evident  that  a 
composite  sample  is  not  what  is  intended,  but  rather  that  each  oyster 
shall  be  examined  separately. 

Some  workers  have  based  their  analysis  upon  several  composite 
samples  of  five  oysters  each,  while  others  have  used  five,  t6n  or  fifteen 
oysters  separately.  There  is  great  variation  in  the  bacterial  content 
of  oysters  from  the  same  lot.  In  one  oyster  there  may  be  one 
hundred  B.  coh  per  cubic  centimeter  of  the  shell  liquor,  while  in 
another  oyster  from  the  same  sample  they  may  be  entirely  absent. 
The  important  consideration  in  the  examination  of  oysters  is  the 
average  number  of  B.  coli  in  the  oysters  as  a  whole  and  not  the  number 
in  any  individual  oyster.  For  this  reason  the  larger  the  sample, 
within  reasonable  limits,  the  more  accurate  the  results  as  an  indication 


BACTERIOLOGY  OF  THE  OYSTER.  85 

of  theB.  coli  content  of  the  oysters  of  any  particular  area.  Smith/ 
in  the  analysis  of  one  hundred  and  twenty-five  oysters  in  each  of  a 
series  of  samples,  came  to  the  conclusion  that  not  less  than  fifteen 
oysters  should  be  used.  The  use  of  too  small  a  sample  may  account 
in  part  for  the  wide  variation  in  results  obtained  by  different  analysts 
in  the  examination  of  the  same  oyster  bed  at  approximately  the  same 
time.  In  the  writer's  opinion  twenty-five  oysters  is  not  too  large  a 
sample  to  be  used  in  any  analysis. 

The  changes  which  the  writer  would  suggest  in  "Standard  Methods 
of  Shellfish  Examination,"  are  as  follows: 

The  size  of  the  sample  should  be  at  least  twenty-five  oysters. 
After  reaching  the  laboratory  the  oysters  should  be  scrubbed 
thoroughly  with  a  stiff  brush  in  water  free  from  B.  coli  and  dried. 
When  ready  for  examination  the  oyster  should  be  held  between  the 
thumb  and  the  fore-finger  and  the  lip  of  the  shell  flamed  in  the  bunsen 
burner  or  burned  off  with  alcohol.  The  opening  should  be  done  with 
an  oyster  knife  which  has  previously  been  burned  with  alcohol.  The 
method  of  drilling  a  hole  through  the  shell  and  pipetting  out  the 
oyster  juice  should  never  be  substituted  as  an  alternative  method. 

The  shell  liquor  of  the  five  oysters  of  each  of  the  five  composite 
samples  should  be  collected  in  sterile,  graduated,  glass-stoppered 
bottles  and  the  bodies  of  the  five  oysters  should  be  placed  in  a  wide- 
mouth,  glass-stoppered  bottle.  The  amount  of  shell  liquor  should 
be  read  off  and  an  equal  amount  of  sterile  one  per  cent,  salt  solution  or 
sea  water  added  to  the  bottle  containing  the  bodies  of  the  oysters. 
The  stopper  should  be  replaced  and  the  bottle  shaken  at  least  one 
hundred  times.  (The  writer's  experience  has  been  that,  if  the  oysters 
are  opened  carefully  so  as  to  avoid  mutilation,  the  bodies  of  the  oysters 
are  damaged  but  very  little  by  this  procedure  unless  the  shaking  is 
especially  vigorous.)  The  salt  solution  and  mucus  should  then  be 
decanted  into  the  bottle  containing  the  shell  liquor  and  the  whole 
shaken  vigorously  one  hundred  times  to  break  up  any  clumps  of 
bacteria  and  to  separate  as  far  as  possible  the  bacteria  from  the  bits 
of  mucus.  The  five  sets  of  oysters  should  be  treated  in  this  manner, 
making  five  samples  of  five  oysters  each.  If  the  operation  is  conducted 
properly  there  should  be  an  equal  quantity  of  shell  lifiuor  and  salt 
solution  in  each  of  the  five  composite  samples. 

^Size  of  the  Sample  Necessary  for  the  Accurate  Determination  of  the  Sanitary  Quality  of  Shell 
Oysters,  American  Journal  of  Public  Health,  HI,  1913,  705. 


86 


BACTERIOLOGY    OF    THE    OYSTER. 


The  subsequent  jirocedure  should  be  the  same  as  that  recom- 
mended by  "Standard  Methods"  and  the  method  of  scoring  should 
be  the  same  except  that  the  score  as  obtained  by  this  method  should 
be  multiplied  by  two,  because  we  are  using  Y2  c-c  1-20  c.c.  and  1-200 
c.c.  of  the  original  shell  liquor  instead  of  1  c.c,  1-10  c.c.  and  1-100  c.c. 
as  recommended  by  "Standard  Methods." 

The  advantages  of  this  method  are  that  we  are  basing  our  exami- 
nation upon  twenty-five  oysters  instead  of  five  and  the  result  will  be 
much  nearer  the  true  bacterial  content  of  the  sample. 

Another  point  worthy  of  consideration  by  the  Committee  on 
"Standard  Methods"  is  the  number  of  bile  tubes  to  be  used  in  the 
different  dilutions.  The  writer  in  all  the  work  reported  in  this  paper 
and  for  a  long  time  previous  has  used  duplicate  tubes.  An  interesting 
feature  of  this  method  is  that  both  tubes  from  each  dilution  show  gas 
only  approximately  two-thirds  of  the  time.  The  writer  has  regarded 
gas  in  either  of  the  two  duplicate  tubes  as  positive  for  the  dilution  and 
has  assigned  it  the  value  as  recommended  by  "Standard  Methods." 
By  using  this  method  approximately  thirty-three  per  cent,  more  B. 
coli  are  found  than  would  be  the  case  if  only  one  tube  were  used. 

"Standard  Methods"  under  "Illustration  of  the  Application  of  the 
Method  of  Rating  Oysters  for  B.  coU"  recommends  the  transferring 
of  a  positive  result  in  a  high  dilution  in  one  oyster  to  a  lower  dilution 
in  another  oyster,  if  in  the  latter  oyster  the  B.  coli  test  is  negative 
in  the  lower  dilution.  Below  is  an  illustrated  case  from  "Standard 
Methods:" 

Case  C.     Results  of  B.  Coli  Tests  in  Dilutions  Indicated. 


Oystek. 

l.Oc.c 

O.lc.c 

O.llc.c 

Numerical 
value. 

1 

2 

+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 
0 

0          [10  (not  100) 
0          ^10 

3 

+          100 

4 

+           10  (not  100) 

5 

0           10  (not  1) 

140  =  rating. 


But  suppose  in  this  sample  the  tubes  have  been  inoculated  in  dupli- 
cate instead  of  one  tube  for  each  dilution.  The  following  table  shows 
a  not  unexpected  result : 


BACTERIOLOGY    OF   THE    OYSTER. 


87 


Results  of  B.  Coli  Test  in  Duplicate  Tubes  in  Dilutions  Indicated. 


Oyster 

1   c.c. 

O.lc.c 

O.OOlc.c 

1 

+     + 
+     + 
+     + 
+     + 
+      0 

+      0 
+      0 

+    + 

+      0 
0       0 

0        0 

2 

0        0 

3 

+       0 
+       0 
0        0 

4 

5    . 

The  question  now  arises  as  to  what  numerical  value  we  shall  assign 
to  the  dilutions  which  are  positive  in  one  tube  and  not  in  the  other. 
Is  it  proper  to  assign  full  value  to  these  dilutions?  Would  it  not  be 
fairer  to  assign  one-half  the  value  recommended  by  "Standard 
Methods"  to  these  dilutions,  because  basing  our  calculation  upon  both 
tubes  there  are  only  one-half  the  B.  coli  present  that  would  be  indicated 
by  the  positive  result  alone? 

Suppose  now  we  wanted  to  transfer  the  positive  result  in  the  1-100 
c.c.  dilution  of  oyster  No.  4.  Should  it  be  transferred  to  the  negative 
tube  in  oyster  No.  5,  or  to  the  1-10  c.c.  dilution  of  the  same  oyster? 
Further,  what  shall  we  do  with  the  positive  result  in  the  1-100  c.c. 
dilution  of  oyster  No.  3.  Shall  it  remain  where  it  is  or  shall  it  be 
transferred  to  the  negative  tube  in  the  1-10  c.c.  dilution  of  oyster 
No.  1  or  2?  Again  suppose  in  oyster  No.  3  in  the  1-100  c.c.  dilution 
both  the  tubes  should  be  positive  and  there  were  only  one  tube  negative 
in  the  1-10  c.c.  dilution  of  any  of  the  oysters,  should  these  two  positive 
tubes  be  separated  and  transferred  to  the  negative  tubes  in  two  of  the 
other  oysters? 

But  whatever  method  we  use  for  transferring,  shall  we  assign  the 
full  value  recommended  by  "Standard  Methods"  to  the  dilutions 
which  are  positive  in  one  dilution  and  negative  in  the  other,  or  shall 
we  assign  the  better  value,  i.  e.,  one-half  the  value  recommended  by 
"  Standard  Methods?  "  By  taking  advantage  of  the  various  possibili- 
ties we  can  obtain  ratings  varying  between  thirty,  the  lowest  possible, 
and  one  hundred  and  forty,  the  highest  possible.  In  the  first  case 
the  oysters  would  be  very  near  the  permissible  standard,  while  in  the 
other,  the  oysters  would  be  considered  badly  polluted.  If  we  use 
three  tubes  in  each  dilution  the  matter  is  still  further  complicated. 


88  BACTERIOLOGY  OF  THE  OYSTER. 

Another  possibility  would  be  to  regard  each  set  of  tubes  separately 
and  average  the  results.  This  would  be  the  simplest  method,  but 
it  would  not  give  so  low  a  result  as  would  be  possible  by  one  of  the 
other  methods.  In  the  case  in  hand  the  rating  would  be  seventy- 
seven  as  against  thirty,  the  rating  obtained  by  one  of  the  other 
methods. 

The  writer  has  a  case  in  mind  in  which  the  rating  on  one  set  of 
tubes  was  three,  which  showed  the  oysters  to  be  in  a  high  state  of 
purity,  while  the  duplicate  set  showed  a  rating  of  thirty-two,  which 
would  condemn  the  oysters  on  the  strict  application  of  the  standard 
set  by  the  Bureau  of  Chemistry.  Obviously  it  would  be  unjust  to 
base  our  rating  on  either  of  the  two  sets  of  tubes  alone. 

In  the  writer's  opinion  the  standard  set  by  the  Bureau  of  Chemistry 
of  twenty-three  as  the  highest  permissible  rating  is  very  stringent 
and  every  opportunity  should  be  given  the  oyster  growers  to  avail 
themselves  of  a  method  of  oyster  analysis  which  will  be  more  accurate 
in  its  results  and  a  method  of  rating  that  will  more  nearly  represent 
the  sanitary  condition  of  their  product. 


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PAT.  JAN.  21,  1908 


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